Detection of chromosomal abnormalities associated with endometrial cancer

ABSTRACT

The methods and compositions described herein address the need for diagnostic method that could be offered to women during yearly checkups to allow for early detection, diagnosis and classification, and treatment of endometrial cancer. In addition, these methods and compositons addresse the current need for improving diagnostic accuracy of biopsy procedures in symptomatic patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.61/395,303, filed May 10, 2010, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the area of detecting,diagnosing, and monitoring of endometrial hyperplasia and carcinoma.

BACKGROUND OF THE INVENTION

Uterine cancer is the fourth most common malignancy diagnosed in womenin the United States (estimated 42,793 cases in 2009) and is the seventhmost common cause of cancer death among U.S. women. Over 95% of alluterine cancers are cancers of the endometrium (lining of the body ofthe uterus). Lifetime probability of developing cancer of the uterus is1 in 40 (U.S.). 35-50% of women ages 35-70 present with one or more riskfactors for endometrial cancer.

Two different clinicopathologic subtypes are recognized based on lightmicroscopic appearance, clinical behavior, and epidemiology: theestrogen-related (type I, endometrioid) and the non-estrogen-relatedtypes (type II, nonendometrioid such as papillary serous and clearcell). Despite it aggressiveness, endometrial cancer is difficult todiagnose, thus many patients present with symptoms of the late-stagecancer.

SUMMARY OF THE INVENTION

In particular embodiments, the present invention provides a method ofdetecting the presence of endometrial carcinoma in a biological samplefrom a subject. The method entails contacting the sample with one ormore probes for one or more chromosome regions selected from the groupconsisting of: 1q, 2p, 2q, 3p, 3q, 7p, 8p, 8q, 9p, 9q, the centromericregion of chromosome 10, 10q, 15q, 16q, 17p, the centromeric region ofchromosome 18, 18q, 19p, 20q, and 22q. The one or more probes areincubated with the sample under conditions in which each probe bindsselectively with a polynucleotide sequence on its target chromosome orchromosomal region to form a stable hybridization complex. Hybridizationof the one or more probes is detected, wherein a hybridization patternshowing at least one gain or loss or imbalance at a chromosomal regiontargeted by the probes is indicative of endometrial carcinoma.

In certain embodiments, a hybridization pattern showing a gain in one ormore chromosome regions selected from the group consisting of: 1q, 2p,3q, 8q, 10q, and 20q is indicative of endometrial carcinoma. In specificembodiments, a hybridization pattern showing a gain in one or morechromosome regions selected from the group consisting of: 1q, 10p, and10q is indicative of endometrioid carcinoma. In other specificembodiments, a hybridization pattern showing a gain in one or morechromosome regions selected from the group consisting of: 3q, 8q, 18q,and 20q is indicative of non-endometrioid carcinoma. In an illustrativeembodiment, a hybridization pattern showing a gain in 1q31-qtel isindicative of endometrial carcinoma.

In certain embodiments, a hybridization pattern showing a loss in one ormore chromosome regions selected from the group consisting of: 9p, 9q,15q, 16q, 17p, 18q, 19p, and 22q is indicative of endometrial carcinoma.In illustrative embodiments, a hybridization pattern showing a loss inone or more chromosome regions selected from the group consisting of:15q11-q13, 18q21, and 19 ptel is indicative of endometrial carcinoma.

In certain embodiments, the one or more probes are for one or morechromosome subregions selected from the group consisting of: 1q25, 2p24,2q26, 3p21, 3q27-q29, 7p21, 8p11, 8q24, 9q34, the centromeric region ofchromosome 10, 10q23, 10q26, 15q11-q13, 16q24, the centromeric region ofchromosome 18, 18q21, 20q12 and 20q13.

In particular embodiments, the sample is contacted with a combination ofat least 3 probes for a set of chromosome subregions selected from thegroup consisting of

-   -   1q25, 8q24, 15q1′-q13;    -   1q25, 10q26, 15q11-q13;    -   1q25, 2p24, 8q24    -   1q25, 8q24, 10q26;    -   1q25, 8p11, 15q11-q13;    -   1q25, 2p24, 8p11;    -   1q25, 8p11, 10q26;    -   1q25, 8p11, 8q24;    -   1q25, 2p24, 10q26;    -   1q25, 2p24, 15q11-q13;    -   8q24, 10q26, 15q11-q13;    -   1q25, 8p11, 20q13;    -   1q25, 8q24, 20q13;    -   1q25, 15q11-q13, 20q13;    -   1q25, 10q26, 20q13;    -   8p11, 10q26, 15q11-q13;    -   1q25, 2p24, 20q13;    -   2p24, 8p11, 10q26;    -   2p24, 8q24, 10q26;    -   2p24, 10q26, 15q11-q13;    -   2p24, 8q24, 15q11-q13;    -   10q26, 15q11-q13, 20q13;    -   1q25, 8p11, 18q21;    -   1q25, 8q24, 18q21;    -   1q25, 10q26, 18q21;    -   1q25, 15q11-q13, 18q21;    -   8p11, 8q24, 10q26;    -   8q24, 10q26, 20q13;    -   1q25, 2p24, 18q21;    -   2p24, 8p11, 8q24;    -   2p24, 8p11, 15q11-q13;    -   8p11, 8q24, 15q11-q13;    -   2p24, 10q26, 20q13;    -   8p11, 10q26, 20q13;    -   2p24, 8p11, 20q13;    -   2p24, 8q24, 20q13;    -   8q24, 15q11-q13, 20q13; and    -   8p11, 15q11-q13, 20q13.

In certain embodiments, the sample is contacted with a combination of atleast 3 probes for a set of chromosome subregions selected from thegroup consisting of:

-   -   1q25, 18q21, CEP18, 8q24;    -   2p24, 2q26, 10q26, 2q13; and    -   10q23, CEP10, and 8p11.

In particular embodiments, the sample is contacted with a combination ofat least 2 probes for a set of chromosome subregions selected from thegroup consisting of:

-   -   18q21, 1q24, 8q24, CEP18;    -   1q24, 8q24, 10q26, CEP18;    -   18q21, 1q24, 10q26, CEP18;    -   1q24, 8q24, CEP18, 3q27-q29;    -   18q21, 1q24, 8q24, 10q26;    -   1q24, 2p24, 10q26, CEP18;    -   1q24, 10q26, CEP18, 3q27-q29;    -   1q24, 10q26, CEP18, 20q13;    -   1q24, CEP18, 3q27-q29, 20q13;    -   1q24, 2p24, CEP18, 3q27-q29;    -   18q21, 1q24, 10q26, 20q13;    -   1q24, 8q24, CEP18;    -   18q21, 1q24, CEP18; and    -   1q24, CEP18.

In illustrative embodiments, the sample is contacted with a combinationof at least 4 probes for a set of chromosome subregions selected fromthe group consisting of:

-   -   1q24, 8q24, CEP18, 20q13;    -   1q24, CEP18, 3q27-q29, 20q13;    -   1q24, CEP18, 20q13, 10q26;    -   CEP10, 8q24, CEP18, 1q24;    -   10q26, CEP10, 1q24, 8q24;    -   8q24, 1q24, 20q13, CEP18; 10q26;    -   20q13, CEP10, 1q24, 10q26,        wherein a hybridization pattern showing a gain in one or more of        these chromosome subregions is indicative of endometrial        carcinoma.

In specific embodiments, one or more of a gain at one of more of 1q24,8q24, CEP18, and 20q13 are indicative of endometrial carcinoma. Inalternative specific embodiments, one or more of a 20q13 gain, a 1q24gain, a CEP10 imbalance, and a 10q26 gain are indicative of endometrialcarcinoma.

In particular embodiments, the sample is contacted with a combination ofat least 2 probes for a set of chromosome subregions selected from thegroup consisting of:

-   -   18q21, 1q25, 8q24, CEP18;    -   1q25, 8q24, 10q26, CEP18;    -   18q21, 1q25, 10q26, CEP18;    -   1q25, 8q24, CEP18, 3q27-q29;    -   18q21, 1q25, 8q24, 10q26;    -   1q25, 2p24, 10q26, CEP18;    -   1q25, 10q26, CEP18, 3q27-q29;    -   1q25, 10q26, CEP18, 20q13;    -   1q25, CEP18, 3q27-q29, 20q13;    -   1q25, 2p24, CEP18, 3q27-q29;    -   18q21, 1q25, 10q26, 20q13;    -   1q25, 8q24, CEP18;    -   18q21, 1q25, CEP18; and    -   1q25, CEP18.

In illustrative embodiments, the sample is contacted with a combinationof at least 4 probes for a set of chromosome subregions selected fromthe group consisting of

-   -   1q25, 8q24, CEP18, 20q13;    -   1q25, CEP18, 3q27-q29, 20q13;    -   1q25, CEP18, 20q13, 10q26;    -   CEP10, 8q24, CEP18, 1q25;    -   10q26, CEP10, 1q25, 8q24;    -   8q24, 1q25, 20q13, CEP18; 10q26;    -   20q13, CEP10, 1q25, 10q26,        wherein a hybridization pattern showing a gain in one or more of        these chromosome subregions is indicative of endometrial        carcinoma.

In specific embodiments, one or more of a gain at one of more of 1q25,8q24, CEP18, and 20q13 are indicative of endometrial carcinoma. Inalternative specific embodiments, one or more of a 20q13 gain, a 1q25gain, a CEP10 imbalance, and a 10q26 gain are indicative of endometrialcarcinoma.

In variations of any of the preceding embodiments, the probe combinationcan distinguish samples including endometrial carcinoma from samplesthat do not include endometrial carcinoma with a sensitivity of at least93% and a specificity of at least 90%. For example, the sensitivity canbe at least 95% and the specificity can be at least 90.4%. In specificembodiments, the sensitivity is least 96% and the specificity is atleast 91%.

In variations of any of the preceding embodiments, the probe combinationcan include between 2 and 10 probes. In particular embodiments, theprobe combination includes between 3 and 8 probes. In an illustrativeembodiment, the probe combination includes 4 probes.

In any of preceding embodiments, the method can be carried out by arraycomparative genomic hybridization (aCGH) to probes immobilized on asubstrate. Alternatively, the method can be carried out by fluorescencein situ hybridization, and each probe in the probe combination can belabeled with a different fluorophore.

In any of the preceding embodiments, the sample can be an endometrialbrushing specimen or an endometrial biopsy specimen.

In any of the preceding claims, when the results of the method indicateendometrial carcinoma, the method can additionally include treating thesubject for endometrial carcinoma.

The invention also provides, in certain embodiments, a combination ofprobes including between 2 and 10 probes selected from any of the groupsset forth above, wherein the combination of probes has a sensitivity ofat least 93% and a specificity of at least 90% for distinguishingsamples including endometrial carcinoma from samples that do not includeendometrial carcinoma. In particular embodiments, the combination ofprobes has a sensitivity of at least 95% and a specificity of at least90.4%. In illustrative embodiments, the combination of probes has asensitivity of at least 96% and a specificity of at least 91%. Invarious embodiments, the probe combination includes between 3 and 8probes, e.g., 4 probes.

Another aspect of the invention includes a kit for diagnosingendometrial carcinoma, wherein the kit includes a combination of probesincluding between 2 and 10 probes selected from any of the groups setforth above, wherein the combination of probes has a sensitivity of atleast 93% and a specificity of at least 90% for distinguishing samplesincluding endometrial carcinoma from samples that do not includeendometrial carcinoma. In particular embodiments, the combination ofprobes has a sensitivity of at least 95% and a specificity of at least90.4%. In illustrative embodiments, the combination of probes has asensitivity of at least 96% and a specificity of at least 91%. Invarious embodiments, the probe combination includes between 3 and 8probes, e.g., 4 probes.

In various embodiments, a chromosomal gain, loss, or imbalance detectedby at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 probes is indicative ofendometrial carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C: The frequency of genomic changes in all cancers from CGHdata.

FIG. 2A(1-3)-B(1-3): The frequency of genomic changes in A, endometrioidcancers; B, non-endometrioid cancers from CGH data.

FIG. 3: Complementation of selected genomic array clones. A, Abnormal(gain or loss); NC, no change in copy number.

FIG. 4A-I: Probe sets 1 and 2 shown on the aCGH data output.

FIG. 5: Representative example of cells with FISH signals(amplification).

FIG. 6A-C: Probe sets 1 and 2. Average % abnormal cells in all specimensevaluated. A, % Cells with Gain; B, % Cells with Loss; C, % Cells withGains and Losses.

FIG. 7: ROC Curve for CEP18+1q24+MYC+DCC, % Abnormal (% cells witheither copy number gain or copy number loss for at least 1 of 4 loci).

FIG. 8: Individual Probes and Combinations (ROC Curves, % Abnormal).

FIG. 9: ROC Curves, 4-Probe Combination, Gains of 1q24, MYC, CEP18 and20q13.2.

FIG. 10: Evaluation of additional probes to improve sensitivity ofendometrial cancer detection.

DETAILED DESCRIPTION

The present invention provides a method of detection of chromosomalabnormalities associated with endometrial carcinoma of both endometrioidand non-endometrioid types, as well as probe combinations and diagnostickits. The methods can utilize techniques known as Comparative GenomicHybridization on a microarray (aCGH) and in situ hybridization (e.g.,Fluorescence In Situ Hybridization (FISH)) using a combination of LocusSpecific Identifier (LSI) and Chromosome Enumerator (CEP) probes todetect cells that have chromosomal abnormalities consistent with adiagnosis of endometrial cancer. The methods described herein can beused to detect endometrial cancer in various types of specimens (e.g.,endometrial brushing specimen or endometrial biopsy specimen) obtainedin the doctors office or operating room.

There is currently no cytological diagnostic test for the earlydetection of endometrial cancer available today, and there is no test orprocedure to routinely screen women at risk for endometrial cancer.Endometrial biopsy is recommended as the initial evaluation of womenwith abnormal uterine bleeding. The disadvantages of biopsy are that itan invasive and uncomfortable procedure for the patient. Moreover,tumors comprising <50% of the endometrium may be inadequately sampled byendometrial biopsy. Inadequate sampling by biopsy may result in falsenegative results and necessitate additional endometrial biopsies todetermine the cause of persistent abnormal uterine bleeding. Asendometrial cancers are relatively fast growing, patients often presentafter the cancers have already developed and spread locally.

Conventional cytology collected with an endometrial sampling device suchas the Tao brush offers the advantage of being relatively non-invasiveand therefore more comfortable for the patient. In addition, endometrialsampling for cytology is less likely than biopsy to result in falsenegative results due to inadequate sampling. The problem withconventional cytology is that most pathologists do not have experiencewith interpreting endometrial cytology and many consider it difficult tointerpret. Furthermore, even experienced cytopathologists find thatthere are significant fraction of cases that cannot be definitelydiagnosed as either positive or negative for cancer and which must becategorized as indeterminate for the presence of cancer.

The methods and compositions described herein will provide means forscreening and improved diagnosis of endometrial cancer. Specifically,the methodology described herein can provide one or more of thefollowing benefits: distinguish cancer from difficult benign conditions;distinguish benign tissue from pre-cancerous lesions and pre-cancerouslesions from cancer; distinguish endometrioid and non-endometrioidtumors; provide an early screening tool for outpatient tests on cytologyspecimens; aid in diagnosis of endometrial cancer in biopsy or surgicalspecimens (aid histological tissue evaluation); and provide an aid inmonitoring of cancer and pre-cancerous conditions during therapy.

Advantages of the methods described herein can include one or more ofthe following: use of stable DNA for detection of chromosomalabnormalities (deletion, amplification, aneusomy, translocation); rapiddetection: results could be obtained in 18-36 hours; implementationpossibilities include multiplexed methods (e.g., microarray) andmulticolor FISH; use as stand-alone test or as adjuncts to other tests(histology, PSA, nomogram, methylation, mutation); use on cytologyspecimens or biopsy (fresh-frozen or FFPE); combination of severalprobes increases sensitivity and specificity as compared to asingle-analyte assay; increased sensitivity compared to conventionalcytology.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “endometrial carcinoma” refers to a malignant neoplasm of theendometrium, which is the mucous membrane lining the uterus. Twodifferent clinicopathologic subtypes are recognized based on lightmicroscopic appearance, clinical behavior, and epidemiology: theestrogen-related (type I, “endometrioid”) and the non-estrogen-relatedtypes (type II, “nonendometrioid”, such as papillary serous and clearcell).

The terms “tumor” or “cancer” in an animal refer to the presence ofcells possessing characteristics such as atypical growth or morphology,including uncontrolled proliferation, immortality, metastatic potential,rapid growth and proliferation rate, and certain characteristicmorphological features. Often, cancer cells will be in the form of atumor, but such cells may exist alone within an animal. The term tumorincludes both benign and malignant neoplasms. The term “neoplastic”refers to both benign and malignant atypical growth.

The term “biological sample” or “specimen” is intended to mean a sampleobtained from a subject suspected of having, or having endometrialcarcinoma. In some embodiments, the sample includes a formalin-fixedparaffin-embedded biopsy. In addition to subjects suspected of havingendometrial carcinoma, the biological sample may further be derived froma subject that has been diagnosed with endometrial carcinoma forconfirmation of diagnosis or establishing that all of the tumor wasremoved (“clear margin”). The sample may be derived from a endometrialbrushing specimen or endometrial biopsy specimen.

The terms “nucleic acid” or “polynucleotide,” as used herein, refer to adeoxyribonucleotide or ribonucleotide in either single- ordouble-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotideswhich have similar or improved binding properties, for the purposesdesired. The term also encompasses nucleic-acid-like structures withsynthetic backbones. DNA backbone analogues provided by the inventioninclude phosphodiester, phosphorothioate, phosphorodithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholinocarbamate, and peptide nucleic acids (PNAs); see Oligonucleotides andAnalogues, a Practical Approach, edited by F. Eckstein, IRL Press atOxford University Press (1991); Antisense Strategies, Annals of the NewYork Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Researchand Applications (1993, CRC Press). PNAs contain non-ionic backbones,such as N-(2-aminoethyl)glycine units. Phosphorothioate linkages aredescribed in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.Pharmacol. 144:189-197. Other synthetic backbones encompassed by theterm include methyl-phosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (Strauss-Soukup (1997)Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag(1996) Antisense Nucleic Acid Drug Dev 6: 153-156).

The terms “hybridizing specifically to,” “specific hybridization,” and“selectively hybridize to,” as used herein, refer to the binding,duplexing, or hybridizing of a nucleic acid molecule preferentially to aparticular nucleotide sequence under stringent conditions. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target sequence, and to a lesser extentto, or not at all to, other sequences. A “stringent hybridization” and“stringent hybridization wash conditions” in the context of nucleic acidhybridization (e.g., as in array, Southern or Northern hybridization, orFISH) are sequence-dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I, Ch. 2, “Overview of principles of hybridization and thestrategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”).Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Verystringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on an array or on a filter in a Southern ornorthern blot is 42° C. using standard hybridization solutions (see,e.g., Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual(3rd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring HarborPress, NY, and detailed discussion, below).

A “chromosomal probe” or “chromosomal probe composition” refers to oneor more polynucleotides that specifically hybridize to a region of achromosome. The target sequences to which the probe can bind vary inlength, e.g., from about 25,000 nucleotides to about 800,000nucleotides. Smaller probes, e.g., that hybridize to a region of lessthan 100,000 nucleotides, or to a region of less than 10,000nucleotides, can also be employed. Thus, in various embodiments, theprobe can hybridize target sequences that are 25,000 nucleotides, 30,000nucleotides, 50,000 nucleotides, 100,000 nucleotides, 150,000nucleotides, 200,000 nucleotides, 250,000 nucleotides, 300,000nucleotides, 350,000 nucleotides, 400,000 nucleotides, 450,000nucleotides, 500,000 nucleotides, 550,000 nucleotides, 600,000nucleotides, 650,000 nucleotides, 700,000 nucleotides, 750,000nucleotides, or 800,00 nucleotides in length or that have a lengthfalling in any range having any of these values as endpoints. A probe toa particular chromosomal region can include multiple polynucleotidefragments, e.g., ranging in size from about 50 to about 1,000nucleotides in length.

A chromosome enumeration probe (CEP) is any probe able to enumerate thenumber of specific chromosomes in a cell.

The term “label containing moiety” or “detection moiety” generallyrefers to a molecular group or groups associated with a chromosomalprobe, either directly or indirectly, that allows for detection of thatprobe upon hybridization to its target.

The term “target region” or “nucleic acid target” refers to a nucleotidesequence that resides at a specific chromosomal location whose lossand/or gain is indicative of the presence of endometrial carcinoma.

INTRODUCTION

The methods described herein are based, in part, on the identificationof highly sensitive and specific chromosomal probe combinations that canbe used to selectively detect endometrial carcinoma. The probecombinations provide higher sensitivity and specificity than individualprobes. The probes encompass locus-specific probes as well as chromosomeenumeration probes (CEPs), which typically hybridize to centromericregions. The methods are carried by hybridizing one or more probes tonucleic acids from, e.g., cytology specimens (uterine brushings,washings, swabs) or cells from frozen specimens or fixed specimens, suchas formalin-fixed, paraffin-embedded tissue.

Chromosomal Probes

Probes for use in the invention are used for hybridization to nucleicacids that are present in biological samples from subjects where thereis some degree of suspicion of endometrial carcinoma. In certainembodiments, the probes are labeled with detectable labels, e.g.,fluorescent labels.

Chromosome Enumeration Probe

A chromosome enumeration probe typically recognizes and binds to aregion near to (referred to as “peri-centromeric”) or at the centromereof a specific chromosome, typically a repetitive DNA sequence. Thecentromere of a chromosome is typically considered to represent thatchromosome entity since the centromere is required for faithfulsegregation during cell division. Deletion or amplification of aparticular chromosomal region can be differentiated from loss or gain ofthe whole chromosome (aneusomy), within which it normally resides, bycomparing the number of signals corresponding to the particular locus(copy number) to the number of signals for the corresponding centromere.One method for making this comparison is to divide the number of signalsrepresenting the locus by the number of signals representing thecentromere. Ratios of less than one indicate relative loss or deletionof the locus, and ratios greater than one indicate relative gain oramplification of the locus. Similarly, comparison can be made betweentwo different loci on the same chromosome, for example on two differentarms of the chromosome, to indicate imbalanced gains or losses withinthe chromosome.

In lieu of a centromeric probe for a chromosome, one of skill in the artwill recognize that a chromosomal arm probe may alternately be used toapproximate whole chromosomal loss or gain. However, such probes are notas accurate at enumerating chromosomes since the loss of signals forsuch probes may not always indicate a loss of the entire chromosomes.Examples of chromosome enumeration probes include CEP® probes (e.g.,CEP® 12 and X/Y probes) commercially available from Abbott Molecular,DesPlaines, Ill. (formerly Vysis, Inc., Downers Grove, Ill.).

Chromosome enumerator probes and locus-specific probes that target achromosome region or subregion can readily be prepared by those in theart or can be obtained commercially, e.g., from Abbott Molecular,Molecular Probes, Inc. (Eugene, Oreg.), or Cytocell (Oxfordshire, UK).Such probes are prepared using standard techniques. Chromosomal probesmay be prepared, for example, from protein nucleic acids, cloned humanDNA such as plasmids, bacterial artificial chromosomes (BACs), and P1artificial chromosomes (PACs) that contain inserts of human DNAsequences. A region of interest may be obtained via PCR amplification orcloning. Alternatively, chromosomal probes may be preparedsynthetically.

Locus-Specific Probes

Probes that can be used in the method described herein include probesthat selectively hybridize to chromosome regions (e.g., 1q, 2p, 2q, 3p,3q, 7p, 8p, 8q, 9p, 9q, 10q, 15q, 16q, 17p, 18q, 19p, 20q, and 22q) orsubregions of the chromosome regions (e.g., 1q25, 2p24, 2q26, 3p21,3q27-q29, 7p21, 8p11, 8q24, 9q34, 10q23, 10q26, 15q11-q13, 16q24, 18q21,20q12 and 20q13). (The subregion designations as used herein include thedesignated band and typically about 10 megabases of genomic sequence toeither side.) Such probes are also referred to as “locus-specificprobes.” A locus-specific probe selectively binds to a specific locus ata chromosomal region that is known to undergo gain or loss inendometrial carcinoma. A probe can target coding or non-coding regions,or both, including exons, introns, and/or regulatory sequences, such aspromoter sequences and the like.

When targeting of a particular gene locus is desired, probes thathybridize along the entire length of the targeted gene are preferred insome embodiments, although not required. In specific embodiments, alocus-specific probe can be designed to hybridize to an oncogene ortumor suppressor gene, the genetic aberration of which is correlatedwith endometrial carcinoma.

Probes useful in the methods described herein generally include acollection of one or more nucleic acid fragments whose hybridization toa target can be detected. Probes can be produced from a source ofnucleic acids from one or more particular (preselected) portions of thegenome, for example one or more clones, an isolated whole chromosome orchromosome fragment, or a collection of polymerase chain reaction (PCR)amplification products. Probes useful in the method described herein canbe produced from nucleic acids found in the regions described herein.The probe may be processed in some manner, for example, by blocking orremoval of repetitive nucleic acids or enrichment with unique nucleicacids.

In certain embodiments, e.g., in in situ hybridization (e.g.,FISH)-based embodiments, locus-specific probe targets preferably includeat least 100,000 nucleotides. For cells of a given sample, relative tothose of a control, increases or decreases in the number of signals fora probe indicate a gain or loss, respectively, for the correspondingregion.

Probes may also be employed as isolated nucleic acids immobilized on asolid surface (e.g., nitrocellulose), as in aCGH. In some embodiments,the probes may be members of an array of nucleic acids as described, forinstance, in WO 96/17958, which is hereby incorporated by reference itits entirety and specifically for its description of array CGH.Techniques capable of producing high density arrays are well-known (see,e.g., Fodor et al. Science 767-773 (1991) and U.S. Pat. No. 5,143,854),both of which are hereby incorporated by reference for this description.

As described in detail below, loci that were putatively associated withendometrial cancer were identified and the sensitivity and specificityof this association was examined in detail using array ComparativeGenomic Hybridization (aCGH) and Fluoresence In Situ Hybridization(FISH). The clones selected from aCGH analysis included: LAMC2(1q25-q31), MYCN (2p24.1), RASSF (3p21.3), TP63 (3q27-q29), IL6 (7p21),FGFR1 (8p11.2-p11.1), MYC (8q24), TSC1 (9q34), PTEN (10q23.3), FGFR2(10q26), UBE3A/D15S10 (15q11-q13), FANCA (16q24.3), DCC (18q21.3), NCOA3(20q12), and ZNF217 (20q13.2). The probes in FISH analysis included:1q25, PTEN (10q23.3), DCC (18q21.2), CEP10, CEP18, FGFR1 (8p11.2), MYC(8q24), MYCN (2p24.3), PIK3CA (2q26.32), FGFR2 (10q26.13), and ZNF217(20q13.2). New FISH probes were developed for NMYC, FGFR1, and FGFR2.

Probe Selection Methods

Probe combinations can be selected for their ability to simply detectendometrial carcinoma, but are typically selected for the ability todiscriminate between endometrial carcinoma and other conditions. Thus,analyses of probe combinations are typically performed to determine theDFI values of different probe combinations for discriminating betweenendometrial carcinoma and other conditions or normal tissue. Inparticular embodiments, probe combinations can be analyzed todiscriminate between endometriod and non-endometrioid types ofendometrial carcinoma.

Probe combinations for use in the methods of the present invention canbe selected using the principles described in the examples. Combinationsof chromosomal probes within a probe combination are chosen forsensitivity, specificity, and detectability regarding endometrialcarcinoma. Sensitivity refers to the ability of a test (e.g. FISH) todetect disease (e.g. endometrial carcinoma) when it is present. Moreprecisely, sensitivity is defined as True Positives/(TruePositives+False Negatives). A test with high sensitivity has few falsenegative results, while a test with low sensitivity has many falsenegative results. In particular embodiments, the combination of probeshas a sensitivity of least about: 93, 94, 95, 96, 97, 98, 99, or 100%,or a sensitivity falling in a range with any of these values asendpoints.

Specificity, on the other hand, refers to the ability of test (e.g.FISH) to give a negative result when disease is not present. Moreprecisely, specificity is defined as True Negatives/(TrueNegatives+False Positives). A test with high specificity has few falsepositive results, while a test with a low specificity has many falsepositive results. In certain embodiments, the combination of probes hasa specificity of at about: 88, 89, 90, 91, 92, 93, 94, or 95%, or aspecificity falling in a range with any of these values as endpoints.

In general, chromosomal probe combinations with the highest combinedsensitivity and specificity for the detection of endometrial carcinomaare preferred. In exemplary embodiments the combination of probes has asensitivity and specificity of at least about: 93% and 88%, 95% and 90%,96% and 91%, 97% and 92%, respectively, or any combination ofsensitivity and specificity based on the values given above for each ofthese parameters.

The combined sensitivity and specificity of a probe combination can berepresented by the parameter distance from ideal (DFI), defined as[(1-sensitivity)²+(1-specificity)²]^(1/2) DFI values range from 0 to1.414, with 0 representing a probe combination having 100% sensitivityand 100% specificity and 1.414 representing a probe combination with 0%sensitivity and 0% specificity.

There is no limit to the number of probes that can be employed in acombination, although, in certain embodiments, no more than ten probesare combined. Additionally, in some embodiments, the number of probeswithin a set that is to be viewed by a human observer (and not withcomputer assisted imaging techniques) may be restricted for practicalreasons, e.g., by the number of unique fluorophores that providevisually distinguishable signals upon hybridization. For example,typically four or five unique fluorophores (e.g., which appear as red,green, aqua, and gold signals to the human eye) can be convenientlyemployed in a single probe combination. Generally, the sensitivity of anassay increases as the number of probes within a set increases. However,the increases in sensitivity become smaller and smaller with theaddition of more probes and at some point the inclusion of additionalprobes to a probe combination is not associated with significantincreases in the sensitivity of the assay (“diminishing returns”).Increasing the number of probes in a probe combination may decrease thespecificity of the assay. Accordingly, a probe combination of thepresent invention typically includes two, three, or four chromosomalprobes, as necessary to provide optimal balance between sensitivity andspecificity.

Individual probes can be chosen for inclusion in a probe combinationbased on their ability to complement other probes within thecombination. Specifically, they are targeted to chromosomes orchromosomal subregions that are not frequently altered simultaneouslywithin a given endometrial carcinoma. Thus, each probe in a probecombination complements the other(s), i.e., identifies endometrialcarcinoma where the other probes in the combination sometime fail toidentify. One method for determining which probes complement one anotheris to identify single probes with the lowest DFI values for a group oftumor specimens. Then additional probes can be tested on the tumorsamples that the initial probe failed to identify, and the probe withthe lowest DFI value measured in combination with the initial probe(s)is added to the set. This may then be repeated until a full set ofchromosomal probes with the desired DFI value is achieved.

Discrimination analysis is one method that can be used to determinewhich probes are best able to detect endometrial carcinoma. This methodassesses if individual probes are able to detect a statisticallydifferent percentage of abnormal cells in test specimens (e.g.,endometrial carcinoma) when compared to normal specimens. The detectionof cells with chromosomal (or locus) gains or chromosomal (or locus)losses can both be used to identify neoplastic cells in endometrialcarcinoma patients. However, chromosomal losses sometimes occur as anartifact in normal cells because of random signal overlap and/or poorhybridization. In sections of formalin-fixed paraffin-embedded material,commonly used to assess biopsies, truncation of nuclei in the sectioningprocess can also produce artifactual loss of chromosomal material.Consequently, chromosomal gains are often a more reliable indicator ofthe presence of neoplastic cells.

Cutoff values for individual chromosomal gains and losses must bedetermined when choosing a probe combination. The term “cutoff value” isintended to mean the value of a parameter associated with chromosomalaberration that divides a population of specimens into two groups—thosespecimens above the cutoff value and those specimens below the cutoffvalue. For example, the parameter may be the absolute number orpercentage of cells in a population that have genetic aberrations (e.g.,losses or gains for target regions). If the number or percentage ofcells in the specimen harboring losses or gains for a particular probeis higher than the cutoff value, the sample is determined to be positivefor endometrial carcinoma.

Useful probe combinations are discussed in detail in the Example below.In exemplary combinations, one or more of a gain at one of more of 1q24,8q24, CEP18, and 20q13 are indicative of endometrial carcinoma, as are(i) one or more of a gain at one of more of 1q25, 8q24, CEP18, and 20q13and (ii) one or more of a 20q13 gain, a 1q24 gain, a CEP10 imbalance,and a 10q26 gain. Also of note are that different genomic changes wereobserved when comparing endometrioid and non-endometrioid subtypes.Gains in chromosomal arms 1q, 10p and 10q were common in endometrioidcarcinomas. Multiple gains across the genome were identified innon-endometrioid carcinomas with the most common gains seen in 3q, 8qand 20q.

Probe Hybridization

Conditions for specifically hybridizing the probes to their nucleic acidtargets generally include the combinations of conditions that areemployable in a given hybridization procedure to produce specifichybrids, the conditions of which may easily be determined by one ofskill in the art. Such conditions typically involve controlledtemperature, liquid phase, and contact between a chromosomal probe and atarget. Hybridization conditions vary depending upon many factorsincluding probe concentration, target length, target and probe G-Ccontent, solvent composition, temperature, and duration of incubation.At least one denaturation step may precede contact of the probes withthe targets. Alternatively, both the probe and nucleic acid target maybe subjected to denaturing conditions together while in contact with oneanother, or with subsequent contact of the probe with the biologicalsample. Hybridization may be achieved with subsequent incubation of theprobe/sample in, for example, a liquid phase of about a 50:50 volumeratio mixture of 2-4.times. SSC and formamide, at a temperature in therange of about 25 to about 55° C. for a time that is illustratively inthe range of about 0.5 to about 96 hours, or more preferably at atemperature of about 32 to about 40° C. for a time in the range of about2 to about 16 hours. In order to increase specificity, use of a blockingagent such as unlabeled blocking nucleic acid as described in U.S. Pat.No. 5,756,696 (the contents of which are herein incorporated byreference in their entirety, and specifically for the description of theuse of blocking nucleic acid), may be used in conjunction with themethods of the present invention. Other conditions may be readilyemployed for specifically hybridizing the probes to their nucleic acidtargets present in the sample, as would be readily apparent to one ofskill in the art.

Upon completion of a suitable incubation period, non-specific binding ofchromosomal probes to sample DNA may be removed by a series of washes.Temperature and salt concentrations are suitably chosen for a desiredstringency. The level of stringency required depends on the complexityof a specific probe sequence in relation to the genomic sequence, andmay be determined by systematically hybridizing probes to samples ofknown genetic composition. In general, high stringency washes may becarried out at a temperature in the range of about 65 to about 80° C.with about 0.2× to about 2×SSC and about 0.1% to about 1% of a non-ionicdetergent such as Nonidet P-40 (NP40). If lower stringency washes arerequired, the washes may be carried out at a lower temperature with anincreased concentration of salt.

Detection of Probe Hybridization Patterns

The hybridization probes can be detected using any means known in theart. Label-containing moieties can be associated directly or indirectlywith chromosomal probes. Different label-containing moieties can beselected for each individual probe within a particular combination sothat each hybridized probe is visually distinct from the others upondetection. Where FISH is employed, the chromosomal probes canconveniently be labeled with distinct fluorescent label-containingmoieties. In such embodiments, fluorophores, organic molecules thatfluoresce upon irradiation at a particular wavelength, are typicallydirectly attached to the chromosomal probes. A large number offluorophores are commercially available in reactive forms suitable forDNA labeling.

Attachment of fluorophores to nucleic acid probes is well known in theart and may be accomplished by any available means. Fluorophores can becovalently attached to a particular nucleotide, for example, and thelabeled nucleotide incorporated into the probe using standard techniquessuch as nick translation, random priming, PCR labeling, and the like.Alternatively, the fluorophore can be covalently attached via a linkerto the deoxycytidine nucleotides of the probe that have beentransaminated. Methods for labeling probes are described in U.S. Pat.No. 5,491,224 and Molecular Cytogenetics: Protocols and Applications(2002), Y.-S. Fan, Ed., Chapter 2, “Labeling Fluorescence In SituHybridization Probes for Genomic Targets,” L. Morrison et al., p. 21-40,Humana Press, both of which are herein incorporated by reference fortheir descriptions of labeling probes.

Exemplary fluorophores that can be used for labeling probes includeTEXAS RED (Molecular Probes, Inc., Eugene, Oreg.), CASCADE blueaectylazide (Molecular Probes, Inc., Eugene, Oreg.), SPECTRUMORANGE™(Abbott Molecular, Des Plaines, Ill.) and SPECTRUMGOLD™ (AbbottMolecular).

One of skill in the art will recognize that other agents or dyes can beused in lieu of fluorophores as label-containing moieties. Suitablelabels that can be attached to probes include, but are not limited to,radioisotopes, fluorophores, chromophores, mass labels, electron denseparticles, magnetic particles, spin labels, molecules that emitluminescence, electrochemically active molecules, enzymes, cofactors,and enzyme substrates. Luminescent agents include, for example,radioluminescent, chemiluminescent, bioluminescent, and phosphorescentlabel containing moieties. Alternatively, detection moieties that arevisualized by indirect means can be used. For example, probes can belabeled with biotin or digoxygenin using routine methods known in theart, and then further processed for detection. Visualization of abiotin-containing probe can be achieved via subsequent binding of avidinconjugated to a detectable marker. The detectable marker may be afluorophore, in which case visualization and discrimination of probesmay be achieved as described above for FISH.

Chromosomal probes hybridized to target regions may alternatively bevisualized by enzymatic reactions of label moieties with suitablesubstrates for the production of insoluble color products. Each probemay be discriminated from other probes within the set by choice of adistinct label moiety. A biotin-containing probe within a set may bedetected via subsequent incubation with avidin conjugated to alkalinephosphatase (AP) or horseradish peroxidase (HRP) and a suitablesubstrate. 5-bromo-4-chloro-3-indolylphosphate and nitro bluetetrazolium (NBT) serve as substrates for alkaline phosphatase, whilediaminobenzidine serves as a substrate for HRP.

In embodiments where fluorophore-labeled probes or probe compositionsare used, the detection method can involve fluorescence microscopy, flowcytometry, or other means for determining probe hybridization. Anysuitable microscopic imaging method may be used in conjunction with themethods of the present invention for observing multiple fluorophores. Inthe case where fluorescence microscopy is employed, hybridized samplesmay be viewed under light suitable for excitation of each fluorophoreand with the use of an appropriate filter or filters. Automated digitalimaging systems such as the MetaSystems, BioView or Applied Imagingsystems may alternatively be used.

In array CGH, the probes are not labeled, but rather are immobilized atdistinct locations on a substrate, as described in WO 96/17958. In thiscontext, the probes are often referred to as the “target nucleic acids.”The sample nucleic acids are typically labeled to allow detection ofhybridization complexes. The sample nucleic acids used in thehybridization may be detectably labeled prior to the hybridizationreaction. Alternatively, a detectable label may be selected which bindsto the hybridization product. In dual- or mult-color aCGH, the targetnucleic acid array is hybridized to two or more collections ofdifferently labeled nucleic acids, either simultaneously or serially.For example, sample nucleic acids (e.g., from endometrial carcinomabiopsy) and reference nucleic acids (e.g., from normal endometrium) areeach labeled with a separate and distinguishable label. Differences inintensity of each signal at each target nucleic acid spot can bedetected as an indication of a copy number difference. Although anysuitable detectable label can be employed for aCGH, fluorescent labelsare typically the most convenient.

Preferred methods of visualizing signals are described in WO 93/18186,which is hereby incorporated by reference for this description. Tofacilitate the display of results and to improve the sensitivity ofdetecting small differences in fluorescence intensity, a digital imageanalysis system can be used. An exemplary system is QUIPS (an acronymfor quantitative image processing system), which is an automated imageanalysis system based on a standard fluorescence microscope equippedwith an automated stage, focus control and filterwheel (Ludl ElectronicProducts, Ltd., Hawthorne, N.Y.). The filterwheel is mounted in thefluorescence excitation path of the microscope for selection of theexcitation wavelength. Special filters (Chroma Technology, Brattleboro,Vt.) in the dichroic block allow excitation of the multiple dyes withoutimage registration shift. The microscope has two camera ports, one ofwhich has an intensified CCD camera (Quantex Corp., Sunnyvale, Calif.)for sensitive high-speed video image display which is used for findinginteresting areas on a slide as well as for focusing. The other cameraport has a cooled CCD camera (model 200 by Photometrics Ltd., Tucson,Ariz.) which is used for the actual image acquisition at high resolutionand sensitivity. The cooled CCD camera is interfaced to a SUN 4/330workstation (SUN Microsystems, Inc., Mountain View, Calif.) through aVME bus. The entire acquisition of multicolor images is controlled usingan image processing software package SCIL-Image (Delft Centre for ImageProcessing, Delft, Netherlands).

Screening and Diagnosis of Patients for Endometrial Carcinoma

The detection methods of the invention include obtaining a biologicalsample from a subject having endometrial carcinoma or suspected ofhaving endometrial carcinoma. The biological sample can be a cytologyspecimen, (e.g, uterine brushing, washing, or swab). In particularembodiments, the biological sample is a frozen or fixed specimen, suchas formalin-fixed and paraffin embedded specimen. The sample iscontacted with one or more chromosomal probe(s) to selectively detectendometrial carcinoma in the sample, if any, under conditions forspecifically hybridizing the probes to their nucleic acid targetspresent in the sample. Probes of a combination can be hybridizedconcurrently or sequentially with the results of each hybridizationimaged digitally, the probe or probes stripped, and the samplethereafter hybridized with the remaining probe or probes. Multiple probecombinations can also be hybridized to the sample in this manner.

The biological sample can be from a patient suspected of havingendometrial carcinoma or from a patient diagnosed with endometrialcarcinoma, e.g., for confirmation of diagnosis or establishing a clearmargin, or for the detection of endometrial carcinoma cells in othertissues such as lymph nodes. The biological sample can also be from asubject with an ambiguous diagnosis in order to clarify the diagnosis.The biological sample can also be from a subject with ahistopathologically benign lesion to confirm the diagnosis. Biologicalsamples can be obtained using any of a number of methods known in theart.

As noted, a biological sample can be treated with a fixative such asformaldehyde and embedded in paraffin and sectioned for use in themethods of the invention. Alternatively, fresh or frozen tissue can bepressed against glass slides to form monolayers of cells known as touchpreparations, which contain intact nuclei and do not suffer from thetruncation artifact of sectioning. These cells may be fixed, e.g., inalcoholic solutions such as 100% ethanol or 3:1 methanol:acetic acid.Nuclei can also be extracted from thick sections of paraffin-embeddedspecimens to reduce truncation artifacts and eliminate extraneousembedded material. Typically, biological samples, once obtained, areharvested and processed prior to hybridization using standard methodsknown in the art. Such processing typically includes protease treatmentand additional fixation in an aldehyde solution such as formaldehyde.

Prescreening of Samples

Prior to detection, cell samples may be optionally pre-selected based onapparent cytologic abnormalities. Pre-selection identifies suspiciouscells, thereby allowing the screening to be focused on those cells.Pre-selection allows for faster screening and increases the likelihoodthat a positive result will not be missed. During pre-selection, cellsfrom a biological sample can be placed on a microscope slide andvisually scanned for cytologic abnormalities commonly associated withdysplastic and neoplastic cells. Such abnormalities includeabnormalities in nuclear size, nuclear shape, and nuclear staining, asassessed by counterstaining nuclei with nucleic acid stains or dyes suchas propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride(DAPI) usually following hybridization of probes to their target DNAs.Typically, neoplastic cells harbor nuclei that are enlarged, irregularin shape, and/or show a mottled staining pattern. Propidium iodide,typically used at a concentration of about 0.4.mu.g/ml to about5.mu.g/ml, is a red-fluorescing DNA-specific dye that can be observed atan emission peak wavelength of 614 nm. DAPI, typically used at aconcentration of about 125 ng/ml to about 1000 ng/ml, is a bluefluorescing DNA-specific stain that can be observed at an emission peakwavelength of 452 nm. In this case, only those cells pre-selected fordetection are subjected to counting for chromosomal losses and/or gains.Preferably, pre-selected cells on the order of at least 20, and morepreferably at least 30-40, in number are chosen for assessingchromosomal losses and/or gains. Preselection of a suspicious region ona tissue section may be performed on a serial section stained byconventional means, such as H&E or PAP staining, and the suspect regionmarked by a pathologist or otherwise trained technician. The same regioncan then be located on the serial section stained by FISH and nucleienumerated within that region. Within the marked region, enumeration maybe limited to nuclei exhibiting abnormal characteristics as describedabove.

Alternatively, cells for detection may be chosen independent ofcytologic or histologic features. For example, all non-overlapping cellsin a given area or areas on a microscope slide may be assessed forchromosomal losses and/or gains. As a further example, cells on theslide, e.g., cells that show altered morphology, on the order of atleast about 50, and more preferably at least about 100, in number thatappear in consecutive order on a microscope slide may be chosen forassessing chromosomal losses and/or gains.

Hybridization Pattern

In Situ Hybridization

The hybridization pattern for the set of chromosomal probes to thetarget regions is detected and recorded for cells chosen for assessmentof chromosomal losses and/or gains. Hybridization is detected by thepresence or absence of the particular signals generated by each of thechromosomal probes. The term “hybridization pattern” is intended torefer to the quantification of chromosomal losses/gains for those cellschosen for such assessment, relative to the number of the same in anevenly matched control sample, for each probe throughout a chosen cellsample. The quantification of losses/gains can include determinationsthat evaluate the ratio of one locus to another on the same or adifferent chromosome. Once the number of target regions within each cellis determined, as assessed by the number of regions showinghybridization to each probe, relative chromosomal gains and/or lossesmay be quantified.

The relative gain or loss for each probe is determined by comparing thenumber of distinct probe signals in each cell to the number expected ina normal cell, i.e., where the copy number should be two. Non-neoplasticcells in the sample, such as keratinocytes, fibroblasts, andlymphocytes, can be used as reference normal cells. More than the normalnumber of probe signals is considered a gain, and fewer than the normalnumber is considered a loss. Alternatively, a minimum number of signalsper probe per cell can be required to consider the cell abnormal (e.g.,5 or more signals). Likewise for loss, a maximum number of signals perprobe can be required to consider the cell abnormal (e.g., 0 signals, orone or fewer signals).

The percentages of cells with at least one gain and/or loss are to berecorded for each locus. A cell is considered abnormal if at least oneof the identified genetic aberrations identified by a probe combinationof the present invention is found in that cell. A sample may beconsidered positive for a gain or loss if the percentage of cells withthe respective gain or loss exceeds the cutoff value for any probes usedin an assay. Alternatively, two or more genetic aberrations can berequired in order to consider the cell abnormal with the effect ofincreasing specificity. For example, wherein gains are indicative of aendometrial carcinoma, a sample is considered positive if it contains,for example, at least four cells showing gains of at least two or moreprobe-containing regions.

aCGH

Array CGH can be carried out in single-color or dual- or multi-colormode. In single-color mode, only the sample nucleic acids are labeledand hybridized to the nucleic acid array. Copy number differences can bedetected by detecting a signal intensity at a particular target nucleicacid spot on the array that differs significantly from the signalintensity observed at one or more spots corresponding to one or moreloci that are present in the sample nucleic acids at a normal copynumber. To facilitate this determination, the array can include targetelements for one or more loci that are not expected to show copy numberdifference(s) in endometrial carcinoma.

In dual- or multi-color mode, signal corresponding to each labeledcollection of nucleic acids (e.g., sample nucleic acids and normal,reference nucleic acids) is detected at each target nucleic acid spot onthe array. The signals at each spot can be compared, e.g., bycalculating a ratio. For example, if the ratio of sample nucleic acidsignal to reference nucleic acid signal exceeds I, this indicates a gainin the sample nucleic acids at the locus corresponding to the targetnucleic acid spot on the array. Conversely, if t if the ratio of samplenucleic acid signal to reference nucleic acid signal is less than 1,this indicates a loss in the sample nucleic acids at the correspondinglocus.

Other Methods of Detecting Copy Number Variations Associated withEndometrial Carcinoma

Those of skill in the art appreciate that copy number variations at anyof the loci described herein can be detected using other methods,including amplification-based methods and high-throughput DNAsequencing.

Amplification-Based Detection

In amplification-based assays, the target nucleic acids act astemplate(s) in amplification reaction(s) (e.g., Polymerase ChainReaction (PCR)). In a quantitative amplification, the amount ofamplification product is proportional to the amount of template in theoriginal sample. Detailed protocols for quantitative PCR are provided inInnis et al. (1990) PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc. N.Y.). A number of commercial quantitative PCRsystems are available, for example the TaqMan system from AppliedBiosystems.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560;Landegren et al. (1988) Science 241: 1077; and Barringer et al. (1990)Gene 89: 117), multiplex ligation-dependent probe amplification (MLPA),transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci.USA 86: 1173), self-sustained sequence replication (Guatelli et al.(1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapterPCR, etc.

Amplification is typically carried out using primers that specificallyamplify one or more loci within each chromosome or chromosomal subregionto be queried. Detection can be carried out by any standard means,including a target-specific probe, a universal probe that binds, e.g.,to a sequence introduced into all amplicons via one or both primers, ora double-stranded DNA-binding dye (such as, e.g., SYBR Green). Inillustrative embodiments, padlock probes or molecular inversion probesare employed for detection.

High-Throughput DNA Sequencing

In particular embodiments, amplification methods are employed to produceamplicons suitable for high-throughput (i.e., automated) DNA sequencing.Generally, amplification methods that provide substantially uniformamplification of target nucleotide sequences are employed in preparingDNA sequencing libraries having good coverage. In the context ofautomated DNA sequencing, the term “coverage” refers to the number oftimes the sequence is measured upon sequencing. The counts obtained aretypically normalized relative to a reference sample or samples todetermine relative copy number. Thus, upon performing automatedsequencing of a plurality of target amplicons, the normalized number oftimes the sequence is measured reflects the number of target ampliconsincluding that sequence, which, in turn, reflects the number of copiesof the target sequence in the sample DNA.

Amplification for sequencing may involve emulsion PCR isolates in whichindividual DNA molecules along with primer-coated beads are present inaqueous droplets within an oil phase. Polymerase chain reaction (PCR)then coats each bead with clonal copies of the DNA molecule followed byimmobilization for later sequencing. Emulsion PCR is used in the methodsby Marguilis et al. (commercialized by 454 Life Sciences), Shendure andPorreca et al. (also known as “Polony sequencing”) and SOLiD sequencing,(developed by Agencourt, now Applied Biosystems). Another method for invitro clonal amplification for sequencing is bridge PCR, where fragmentsare amplified upon primers attached to a solid surface, as used in theIllumina Genome Analyzer. Some sequencing methods do not requireamplification, for example, the single-molecule method developed by theQuake laboratory (later commercialized by Helicos). This method usesbright fluorophores and laser excitation to detect pyrosequencing eventsfrom individual DNA molecules fixed to a surface. Pacific Bioscienceshas also developed a single molecule sequencing approach that does notrequire amplification.

After in vitro clonal amplification (if necessary), DNA molecules thatare physically bound to a surface are sequenced. Sequencing bysynthesis, like dye-termination electrophoretic sequencing, uses a DNApolymerase to determine the base sequence. Reversible terminator methods(used by Illumina and Helicos) use reversible versions ofdye-terminators, adding one nucleotide at a time, and detectfluorescence at each position in real time, by repeated removal of theblocking group to allow polymerization of another nucleotide.Pyrosequencing (used by 454) also uses DNA polymerization, adding onenucleotide species at a time and detecting and quantifying the number ofnucleotides added to a given location through the light emitted by therelease of attached pyrophosphates.

Pacific Biosciences Single Molecule Real Time (SMRT™) sequencing relieson the processivity of DNA polymerase to sequence single molecules anduses phospholinked nucleotides, each type labeled with a differentcolored fluorophore. As the nucleotides are incorporated into acomplementary DNA strand, each is held by the DNA polymerase within adetection volume for a greater length of time than it takes a nucleotideto diffuse in and out of that detection volume. The DNA polymerase thencleaves the bond that previously held the fluorophore in place and thedye diffuses out of the detection volume so that fluorescence signalreturns to background. The process repeats as polymerization proceeds.

Sequencing by ligation uses a DNA ligase to determine the targetsequence. Used in the Polony method and in the SOLiD technology, thismethod employs a pool of all possible oligonucleotides of a fixedlength, labeled according to the sequenced position. Oligonucleotidesare annealed and ligated; the preferential ligation by DNA ligase formatching sequences results in a signal informative of the nucleotide atthat position.

Probe Combinations and Kits for Use in Diagnostic and/or PrognosticApplications

The invention includes highly specific and sensitive combinations ofprobes, as described herein, that can be used to detect endometrialcarcinoma and kits for use in diagnostic, research, and prognosticapplications. Kits include probe combinations and can also includereagents such as buffers and the like. The kits may includeinstructional materials containing directions (i.e., protocols) for thepractice of the methods of this invention. While the instructionalmaterials typically include written or printed materials they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated by this invention.Such media include, but are not limited to electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,CD ROM), and the like. Such media may include addresses to internetsites that provide such instructional materials.

REFERENCES

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All publications cited herein are explictly incorporated by reference.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Array CGH Studies

Study Design

In a collaborative study of Abbott Molecular and Mayo Clinic carried outin 2007-2009, fresh-frozen, biopsy from proven endometrial carcinomaspecimens from 62 patients were obtained with informed consent at thetime of surgery from Rochester Methodist Hospital at Mayo Clinic,Rochester, Minn. Serial 5-μm sections were cut from the collected tissueand sections were stained with hematoxylin and eosin (H&E) forhistological analysis. Specimens with greater than 70% tumor wereselected for aCGH analysis and remaining sections were left unstainedfor future clinical studies.

DNA from the selected specimens was extracted and amplified prior to CGHanalysis by the GenoSensor™ system. Vysis GenoSensor DNA microarrayslides obtained from Abbott Molecular (Des Plaines, Ill.) were used forCGH analysis. The microarray slides contained 287 DNA targets consistingof known oncogenes, tumor suppressor genes, and regions of gain, loss,or loss of heterozygosity commonly associated with cancer (Attachment1). The targets were arrayed in triplicate from BAC libraries.

Test and normal reference DNA samples were random-prime labeled, usingVysis GeneSensor labeling reagents, with Cyanine-3-dCTP, andCyanine-5-dCTP (Perkin Elmer/NEN), respectively. Random priming refersto the process whereby synthetic DNA octamers of random sequence bind tocomplementary DNA sequences (along a DNA template) and serve astemplates for DNA synthesis and elongation by DNA Polymerase I (KlenowFragment). Following additional purification, test and reference DNAwere mixed in equal proportion (in hybridization buffer), denatured, andhybridized against the Vysis GenoSensor™ Array 300 human genomic DNAmicroarray. Hybridization proceeded at 30° C. for 72 hours, followed bywashing and scanning of arrays. Array images were analyzed withGenoSensor™ software, which segments and identifies each target usingthe blue (DAPI) image plane. The software measures mean intensities fromthe green and red image planes, subtracts background, determines meanratio of green/red signal, and calculates the ratio most representativeof the modal DNA copy number of the sample DNA. Gender mismatched(male/female or female/male) hybridizations provided control withrespect to the detection of autosomal copy number imbalances. Arrayswere hybridizes at Mayo Clinic and aCGH data obtained was analyzed byAbbott Molecular.

In total, data for 57 Cancer specimens (44 endometrioid and 13non-endometrioid) and 9 normal specimens was available for analysis.

Results

The CGH data revealed several promising genomic targets for theseinvestigational FISH probe sets (FIG. 1). The most frequent gainsobserved in all endometrial carcinomas included areas on chromosomalarms 1q, 2p, 3q, 8q, and 20q. Most common losses seen in all carcinomasincluded 9p, 9q, 16q, 17p, 18q, and 22q. Interestingly, differentgenomic changes were observed when comparing endometrioid andnon-endometrioid subtypes. Gains in chromosomal arms 1q, 10p and 10qwere common in endometrioid carcinomas. Multiple gains across the genomewere identified in non-endometrioid carcinomas with the most commongains seen in 3q, 8q and 20q.

Gross analysis of chromosomal changes demonstrated that most frequentchanges, of all cancers analyzed, were as follows:

1. Gains on 1q31-qtel (20-30%)

2. Gains on 3q, 8q and 10q (about 20%)

3. Gain on 2p (about 15%)

4. Losses on 9q (about 18%)

5. Losses on 15q11-q13 (about 18%)

6. Losses on 18q21.3 and 19 ptel (about 18-19%)

7. Losses 16q and 17p (about 12%)

Table 1 provides details of the most frequently affected loci (by arrayclone).

TABLE 1 Loci with the frequently of genomic copy number imbalanceof >10% in cancer specimens. FREQ FREQ FREQ FREQ FREQ FREQ FREQ FREQFREQ GAIN LOSS IMBAL GAIN LOSS GAIN LOSS Clone Cyto GAIN LOSS Ca Ca CaEN EN NE NE # Clone Name Location (%) (%) (%) (%) (%) (%) (%) (%) (%) 8AMC2 q25-q31 7.27 .00 1.58 .00 1.58 4.09 .00 3.08 .00 0 GFB2 q41 2.73.00 6.32 .00 6.32 1.82 .00 .69 .00 7 I-5663, q21 1.21 .00 4.56 .00 4.567.27 .00 5.38 .00 I-13414 1 KT3 q44 2.73 .00 4.56 .00 4.56 9.55 .00 .69.00 2 HGC-18290 q tel 2.73 .00 4.56 .00 4.56 9.55 .00 .69 .00 3629F16/SP6 9p tel .03 6.67 .51 9.30 2.81 .27 8.18 .69 3.08 3 QTEL10 q tel6.67 .00 9.30 .00 9.30 5.00 .00 .00 .00 6 YCN(N-myc) p24.1 2.12 .55 4.04.26 9.30 .82 .82 8.46 .00 4 FI2 q tel 6.67 .00 9.30 .00 9.30 1.36 .006.15 .00 10 YC q24.12- 6.67 .00 9.30 .00 9.30 .82 .00 1.54 .00 q24.13 11TK2 q24-qter 6.67 .00 9.30 .00 9.30 .82 .00 1.54 .00 22 SC1 q34 .52 5.15.75 7.54 9.30 .27 .09 .00 6.15 35 GFR2 0q26 6.67 .00 9.30 .00 9.30 0.45.00 5.38 .00 82 BE3A, 5q11-q13 .52 5.15 .75 7.54 9.30 .27 3.64 .00 0.7715S10 31 CC 8q21.3 .52 6.67 .75 7.54 9.30 .27 .82 .00 3.85 3 P63 q27-q295.15 .00 7.54 .00 7.54 .09 .00 6.15 .00 06 GFR1 p11.2- 2.12 .03 4.04 .517.54 .09 .27 0.77 .69 p11.1 21 BCCR1 q33.2 .52 3.64 .75 5.79 7.54 .27.82 .00 6.15 6 QTEL11 q tel 0.61 .55 0.53 .26 5.79 3.64 .27 .00 5.38 028S596 p tel .58 .06 .77 .02 5.79 .82 .55 5.38 5.38 96 RA16D 6q23.2 .030.61 .51 2.28 5.79 .00 .09 5.38 3.08 70 F2 2q12.2 .58 .06 .77 .02 5.79.82 .82 5.38 .69 71 DGFB(SIS) 2q13.1 .69 .15 .77 .02 5.79 .82 .27 5.383.08 9 TGS2(COX2) q31.1 3.64 .00 5.79 .00 5.79 0.45 .00 .00 .00 7 SH2,KCNK12 p22.3- 3.64 .00 5.79 .00 5.79 3.64 .00 3.08 .00 2p22.1 09 XT1q24.11- 3.64 .00 5.79 .00 5.79 .82 .00 6.15 .00 q24.13 12 HGC-3110 q tel3.64 .00 5.79 .00 5.79 .55 .00 3.85 .00 6 1S2465, p12 0.61 .52 2.28 .754.04 .82 .27 0.77 .00 1S3402 99 ANCA 6q24.3 .52 0.61 .75 2.28 4.04 .27.09 .00 3.08 0 L6 p21 2.12 .00 4.04 .00 4.04 1.36 .00 3.08 .00 01 8S504p tel .06 .06 .02 .02 4.04 .82 .55 .69 5.38 37 tSG27915 0q tel 2.12 .004.04 .00 4.04 5.91 .00 .69 .00 97 DH13 6q24.2- .00 2.12 .00 4.04 4.04.00 .09 .00 0.77 q24.3 5 PTEL27 p tel 0.61 .00 2.28 .00 2.28 1.36 .005.38 .00 8 2S447 q tel .06 .55 .02 .26 2.28 .09 .27 .00 5.38 5 ASSF1p21.3 0.61 .00 2.28 .00 2.28 .09 .00 3.08 .00 7 4S2930 q tel .03 .58 .51.77 2.28 .55 .27 .00 0.77 8 84C11/T3 p tel .09 .52 0.53 .75 2.28 .09 .005.38 .69 2 TR1B q13 .09 .52 0.53 .75 2.28 .09 .27 5.38 .00 36 MBT10q25.3- .09 .52 0.53 .75 2.28 3.64 .00 .00 .69 q26.1 78 GH(D14S308) 4qtel 2.12 .00 2.28 .00 2.28 .09 .00 3.08 .00 08 17S125, 7p12-p11.2 .000.61 .00 2.28 2.28 .00 .55 .00 8.46 17S61 10 LGL1 7p12- .00 0.61 .002.28 2.28 .00 .82 .00 0.77 17p11.2 48 OP1 0q12-q13.1 .09 .52 0.53 .752.28 .55 .27 0.77 .00 49 COA3(AIB1) 0q12 0.61 .00 2.28 .00 2.28 .27 .006.15 .00 57 PD52L2, TOM 0q tel 0.61 .00 2.28 .00 2.28 .55 .00 8.46 .0069 CR 2q11.23 .55 .58 .51 .77 2.28 .00 .82 5.38 5.38

As evident from FIG. 2, Non-Endometrioid (NE) tumors had overall greaternumber of genomic changes as compared to Endometrioid (EN) tumors. Inaddition, the pattern of changes was different between EN and NE tumors.For example, changes at 1q and 10q loci were more prevalent in ENtumors, while changes at 8q and 18q appeared more prevalent in NEtumors.

Sensitivity and Specificity Analysis

Analysis was carried out to determine changes in which loci are mostabundant in cancers (both NE and EN) and give the highest sensitivityand specificity in detecting cancer.

First, analysis has been carried out on individual loci and the lociwith the best sensitivity and specificity value as represented by theDFI parameter calculated as:

DFI=√{square root over ((1−SENS)²+(1−SPEC)²)}{square root over((1−SENS)²+(1−SPEC)²)}

were selected. As shown below, the best performance was demonstrated byloci located on the long arm of chromosome 1.

case # SPEC vs DFI vs PROBE 1 specimens SENS norm norm LAMC2:18:1q25-q3157 0.3158 1.0000 0.6842 TGFB2:20:1q41 57 0.2632 1.0000 0.7368 WI-5663,WI- 57 0.2456 1.0000 0.7544 13414:17:1q21 SHGC-18290:22:1q tel 57 0.24561.0000 0.7544 AKT3:21:1q44 57 0.2456 0.8889 0.7625

Then, we considered additional clones, and chosen a representative probefor a segment where several probes were located in one apparentcontiguous region of rearrangement. It is evident (Table 2) that thesensitivity and specificity of the individual loci is low. Therefore,combinations of the loci listed below were evaluated.

TABLE 2 Individual clone performance for selected clones. control case ## Case Control speci- speci- SPEC DFI marker marker marker marker Clonemens SENS mens SENS vs norm vs norm (s)+ (s)− (s)+ (s)− chi sq probLAMC2:18:1q25-q31 abnorm 9 0.0000 57 0.3158 1.0000 0.6842 18 39 0 94.8060E−02 MYCN(N-myc):26:2p24.1 abnorm 9 0.0000 57 0.1930 1.0000 0.807011 46 0 9 1.4883E−01 RASSF1:45:3p21.3 abnorm 9 0.0000 57 0.1228 1.00000.8772 7 50 0 9 2.6617E−01 TP63:53:3q27-q29 abnorm 9 0.0000 57 0.17541.0000 0.8246 10 47 0 9 1.7252E−01 IL6:90:7p21 abnorm 9 0.0000 57 0.14041.0000 0.8596 8 49 0 9 2.3056E−01 FGFR1:106:8p11.2-p11.1 abnorm 9 0.000057 0.1754 1.0000 0.8246 10 47 0 9 1.7252E−01 MYC:110:8q24.12-q24.13abnorm 9 0.0000 57 0.1930 1.0000 0.8070 11 46 0 9 1.4883E−01TSC1:122:9q34 abnorm 9 0.0000 57 0.1930 1.0000 0.8070 11 46 0 91.4883E−01 PTEN:134:10q23.3 abnorm 9 0.2222 57 0.0702 0.7778 0.9560 4 532 7 1.4034E−01 FGFR2:135:10q26 abnorm 9 0.0000 57 0.1930 1.0000 0.807011 46 0 9 1.4883E−01 UBE3A, D15S10:182:15q11-q13 abnorm 9 0.0000 570.1930 1.0000 0.8070 11 46 0 9 1.4883E−01 FANCA:199:16q24.3 abnorm 90.0000 57 0.1404 1.0000 0.8596 8 49 0 9 2.3056E−01 DCC:231:18q21.3abnorm 9 0.1111 57 0.1930 0.8889 0.8146 11 46 1 8 5.5398E−01NCOA3(AIB1):249:20q12 abnorm 9 0.0000 57 0.1228 1.0000 0.8772 7 50 0 92.6617E−01 ZNF217(ZABC1):254:20q13.2 abnorm 9 0.0000 57 0.0702 1.00000.9298 4 53 0 9 4.1224E−01

Complementation between the selected loci, in all of the testedspecimens is represented in FIG. 3.

Sensitivity and specificity in tumor detection was then evaluated usingJMP 8.0 statistical analysis software (SAS Institute), utilizing Fit Xby Y contingency table analysis. In this analysis, all 13 loci, andgroups of 10, 9 and 8 complimentary clones were evaluated. A sample wascalled positive when either one of the loci (at least one locus) in thegroup was positive.

1. A set of all 14 clones: Pos/Neg 14 Loci Row % NEG POS Tumor

NORMAL 88.89 11.11 TUMOR 28.07 71.93 Tests N DF −LogLike RSquare (U) 9901 94.293904 0.1453 Test ChiSquare Prob > ChiSq Likelihood Ratio 188.588<.0001* Pearson 186.366 <.0001* Fisher's Exact Test Prob AlternativeHypothesis Left 1.0000 Prob(Pos/Neg 14 Loci = POS) is greater for Tumor?= NORMAL than TUMOR Right <.0001* Prob(Pos/Neg 14 Loci = POS) is greaterfor Tumor? = TUMOR than NORMAL 2-Tail <.0001* Prob(Pos/Neg 14 Loci =POS) is different across Tumor? 2. A representative subset of 10 clones:DCC FGFR2 IL6 LAMC2 MYC MYCN(N-myc) PTEN RASSF1 TSC1 UBE3A, D15S10Pos/Neg 10 Row % NEG POS

umor? NORMAL 66.67 33.33 TUMOR 26.32 73.68 Tests N DF −LogLike RSquare(U) 924 1 37.838597 0.0655 Test ChiSquare Prob > ChiSq Likelihood Ratio75.677 <.0001* Pearson 81.670 <.0001* Fisher's Exact Test ProbAlternative Hypothesis Left 1.0000 Prob(Pos/Neg 10 = POS) is greater forTumor? = NORMAL than TUMOR Right <.0001* Prob(Pos/Neg 10 = POS) isgreater for Tumor? = TUMOR than NORMAL 2-Tail <.0001* Prob(Pos/Neg 10 =POS) is different across Tumor? 3. A representative subset of 9 clonesDCC FGFR2 IL6 LAMC2 MYC MYCN(N-myc) RASSF1 TSC1 UBE3A, D15S10 Pos/Neg 9Row % NEG POS Tumor

NORMAL 88.89 11.11 TUMOR 28.07 71.93 Tests N DF −LogLike RSquare (U) 9241 88.007643 0.1453 Test ChiSquare Prob > ChiSq Likelihood Ratio 176.015<.0001* Pearson 173.942 <.0001* Fisher's Exact Test Prob AlternativeHypothesis Left 1.0000 Prob(Pos/Neg 9 = POS) is greater for Tumor? =NORMAL than TUMOR Right <.0001* Prob(Pos/Neg 9 = POS) is greater forTumor? = TUMOR than NORMAL 2-Tail <.0001* Prob(Pos/Neg 9 = POS) isdifferent across Tumor? 4. A representative subset of 8 clones FGFR2 IL6LAMC2 MYC MYCN(N-myc) RASSF1 TSC1 UBE3A, D15S10 Pos/Neg 8 Row % NEG POS

umor? NORMAL 100.0 0.00 TUMOR 29.82 70.18 Tests N DF −LogLike RSquare(U) 924 1 133.24371 0.2151 Test ChiSquare Prob > ChiSq Likelihood Ratio266.487 <.0001* Pearson 224.453 <.0001* Fisher's Exact Test ProbAlternative Hypothesis Left 1.0000 Prob(Pos/Neg 8 = POS) is greater forTumor? = NORMAL than TUMOR Right <.0001 * Prob(Pos/Neg 8 = POS) isgreater for Tumor? = TUMOR than NORMAL 2-Tail <.0001 * Prob(Pos/Neg 8 =POS) is different across Tumor?

indicates data missing or illegible when filed

FISH Probe Set Selection

To select 4-color probe sets for further FISH experiments, we evaluatedthe selected loci in greater detail supplementing statistical analysiswith literature review. From the further analysis, for the probeselection, we excluded loci that were previously implicated in benignendometrial diseases. In addition, we considered NCOA3 and ZNF217 to bepotentially located on one segment of rearrangement and thus pickedavailable probe, ZNF 217, for further FISH studies.

Clone on array: Vysis FISH probe availability: 1q25-q31, LAMC2 1q25probe 18q21.3, DCC: loss probe available 1q24, MYC probe available10q26, FGFR2: gain (10q23 in no probe/clone PTENq23.3 literature) 15q11,UBE3A: deletion PWS probe 3q27-q29, TP63: gain (3q24-26.4 2 clones orsubstitute by PIK3CA reported in literature) 3p21.3, RASSF1: gain 2clones 7p21, IL6: gain 1 clone 2p24.1, MYCN: gain SG, SO probes 8p11.2,FGFR1: gain/deletion 2 clones 9q33-34 (TSC1): loss (found inendometriosis also) 20q12-q13 (NCOA3?): gain (in 20q11.2 HIRA, 20q13.2ZNF217 literature q13.2) 16q24.2-q24.3: loss (16q literature)

As evident from the results outlined above, CGH data produced by theGenosenor array yielded several preliminary chromosomal targets whichincluded LAMC2 (1q25), NMYC (2p24.1), PIK3CA (3q27-q29), MYC (8q24),FGFR2 (10q26), centromeric region of chromosome 18 (CEP18), DCC (18q21),and ZNF217 (20q13). Of these eight probes, numerous potential four-probecombinations have been identified and probes were grouped in probe sets.

Contingency table analysis was used to assess probe combinations, andthe combinations were ranked by a chi square p value and by the DFIvalue. As at least some of the loci (such as DCC) represent deletions,we first assessed 3-probe combinations to allow one ChromosomeEnumerator Probe (CEP) to be added to the probe set if required, as acontrol for a deletion probe. Shown below is the list of best 3-probecombination in detecting endometrial cancer (Table 3) with chi square pvalue of <0.05.

The following probe sets were selected and used in FISH experimentsusing Table 3 above as a guide. New probes were designed andmanufactured in Abbott Molecular R&D as indicated below.

Probe Set #1 1q25 SpectrumGold DCC (18q21.2) SpectrumRed CEP18SpectrumGreen MYC (8q24) SpectrumAqua Probe Set #2 MYCN (2p24.3)SpectrumGreen (new probe) PIK3CA (2q26.32) SpectrumGold FGFR2 (10q26.13)SpectrumAqua (new probe) ZNF217 (20q13.2) pectrumRed Probe Set #3 PTEN(10q23.3) SpectrumOrange CEP10 SpectrumGreen FGFR1 (8p11.2) SpectrumAqua(new probe)

TABLE 3 Selected best 3-probe combinations (p < 0.05) Normal Cancer # #spec- marker marker # spec- PROBE 1 PROBE 2 PROBE 3 Probes imens SENS(s)+ (s)− imens LAMC2:18:1q25-q31 MYC:110:8q24.12-q24.13 UBE3A,D15S10:182:15q11- 3 9 0.0000 0 9 57 LAMC2:18:1q25-q31 FGFR2:135:10q26UBE3A, D15S10:182:15q11- 3 9 0.0000 0 9 57 LAMC2:18:1q25-q31MYCN(N-myc):26:2p24.1 MYC:110:8q24.12-q24.13 3 9 0.0000 0 9 57LAMC2:18:1q25-q31 MYC:110:8q24.12-q24.13 FGFR2:135:10q26 3 9 0.0000 0 957 LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1 UBE3A, D15S10:182:15q11- 3 90.0000 0 9 57 LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1FGFR1:106:8p11.2-p11.1 3 9 0.0000 0 9 57 LAMC2:18:1q25-q31FGFR1:106:8p11.2-p11.1 FGFR2:135:10q26 3 9 0.0000 0 9 57LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1 MYC:110:8q24.12-q24.13 3 90.0000 0 9 57 LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1 FGFR2:135:10q26 39 0.0000 0 9 57 LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1 UBE3A,D15S10:182:15q11- 3 9 0.0000 0 9 57 MYC:110:8q24.12-q24.1FGFR2:135:10q26 UBE3A, D15S10:182:15q11- 3 9 0.0000 0 9 57LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1 ZNF217(ZABC1):254:20q13 3 90.0000 0 9 57 LAMC2:18:1q25-q31 MYC:110:8q24.12-q24.132NF217(ZABC1):254:20q13 3 9 0.0000 0 9 57 LAMC2:18:1q25-q31 UBE3A,D15S10:182:15q11 ZNF217(ZABC1):254:20q13 3 9 0.0000 0 9 57LAMC2:18:1q25-q31 FGFR2:135:10q26 ZNF217(ZABC1):254:20q13 3 9 0.0000 0 957 FGFR1:106:8p11.2-p11 FGFR2:135:10q26 UBE3A, D15S10:182:15q11- 3 90.0000 0 9 57 LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1ZNF217(ZABC1):254:20q13 3 9 0.0000 0 9 57 MYCN(N-myc):26:2p24.FGFR1:106:8p11.2-p11.1 FGFR2:135:10q26 3 9 0.0000 0 9 57MYCN(N-myc):26:2p24. MYC:110:8q24.12-q24.13 FGFR2:135:10q26 3 9 0.0000 09 57 MYCN(N-myc):26:2p24. FGFR2:135:10q26 UBE3A, D15S10:182:15q11- 3 90.0000 0 9 57 MYCN(N-myc):26:2p24. MYC:110:8q24.12-q24.13 UBE3A,D15S10:182:15q11- 3 9 0.0000 0 9 57 FGFR2:135:10q26 UBE3A,D15S10:182:15q11 ZNF217(ZABC1):254:20q13 3 9 0.0000 0 9 57LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1 DCC:231:18q21.3 3 9 0.1111 1 857 LAMC2:18:1q25-q31 MYC:110:8q24.12-q24.13 DCC:231:18q21.3 3 9 0.1111 18 57 LAMC2:18:1q25-q31 FGFR2:135:10q26 DCC:231:18q21.3 3 9 0.1111 1 8 57LAMC2:18:1q25-q31 UBE3A, D15S10:182:15q11 DCC:231:18q21.3 3 9 0.1111 1 857 FGFR1:106:8p11.2-p11. MYC:110:8q24.12-q24.13 FGFR2:135:10q26 3 90.0000 0 9 57 MYC:110:8q24.12-q24.1 FGFR2:135:10q26ZNF217(ZABC1):254:20q13 3 9 0.0000 0 9 57 LAMC2:18:1q25-q31MYCN(N-myc):26:2p24.1 DCC:231:18q21.3 3 9 0.1111 1 8 57MYCN(N-myc):26:2p24. FGFR1:106:8p11.2-p11.1 MYC:110:8q24.12-q24.13 3 90.0000 0 9 57 MYCN(N-myc):26:2p24. FGFR1:106:8p11.2-p11.1 UBE3A,D15S10:182:15q11- 3 9 0.0000 0 9 57 FGFR1:106:8p11.2-p11.MYC:110:8q24.12-q24.13 UBE3A, D15S10:182:15q11- 3 9 0.0000 0 9 57MYCN(N-myc):26:2p24. FGFR2:135:10q26 ZNF217(ZABC1):254:20q13 3 9 0.00000 9 57 FGFR1:106:8p11.2-p11. FGFR2:135:10q26 ZNF217(ZABC1):254:20q13 3 90.0000 0 9 57 MYCN(N-myc):26:2p24. FGFR1:106:8p11.2-p11.1ZNF217(ZABC1):254:20q13 3 9 0.0000 0 9 57 MYCN(N-myc).26:2p24.MYC:110:8q24.12-q24.13 ZNF217(ZABC1):254:20q13 3 9 0.0000 0 9 57MYC:110:8q24.12-q24.1 UBE3A, D15S10:182:15q11 ZNF217(ZABC1):254:20q13 39 0.0000 0 9 57 FGFR1:106:8p11.2-p11. UBE3A, D15S10:182:15q11ZNF217(ZABC1):254:20q13 3 9 0.0000 0 9 57 Cancer SPEC DFI vs markermarker chi sq PROBE 1 PROBE 2 PROBE 3 SENS vs norm norm (s)+ (s)− probLAMC2:18:1q25-q31 MYC:110:8q24.12-q24.13 UBE3A, D15S10:182:15q11- 0.54391.0000 0.4561 31 26 0.0024 LAMC2:18:1q25-q31 FGFR2:135:10q26 UBE3A,D15S10:182:15q11- 0.5439 1.0000 0.4561 31 26 0.0024 LAMC2:18:1q25-q31MYCN(N-myc):26:2p24.1 MYC:110:8q24.12-q24.13 0.5263 1.0000 0.4737 30 270.0032 LAMC2:18:1q25-q31 MYC:110:8q24.12-q24.13 FGFR2:135:10q26 0.52631.0000 0.4737 30 27 0.0032 LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1UBE3A, D15S10:182:15q11- 0.5263 1.0000 0.4737 30 27 0.0032LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1 FGFR1:106:8p11.2-p11.1 0.50881.0000 0.4912 29 28 0.0043 LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1FGFR2:135:10q26 0.5088 1.0000 0.4912 29 28 0.0043 LAMC2:18:1q25-q31FGFR1:106:8p11.2-p11.1 MYC:110:8q24.12-q24.13 0.4912 1.0000 0.5088 28 290.0056 LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1 FGFR2:135:10q26 0.49121.0000 0.5088 28 29 0.0056 LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1UBE3A, D15S10:182:15q11- 0.4737 1.0000 0.5263 27 30 0.0072MYC:110:8q24.12-q24.1 FGFR2:135:10q26 UBE3A, D15S10:182:15q11- 0.47371.0000 0.5263 27 30 0.0072 LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1ZNF217(ZABC1):254:20q13 0.4737 1.0000 0.5263 27 30 0.0072LAMC2:18:1q25-q31 MYC:110:8q24.12-q24.13 2NF217(ZABC1):254:20q13 0.47371.0000 0.5263 27 30 0.0072 LAMC2:18:1q25-q31 UBE3A, D15S10:182:15q11ZNF217(ZABC1):254:20q13 0.4737 1.0000 0.5263 27 30 0.0072LAMC2:18:1q25-q31 FGFR2:135:10q26 ZNF217(ZABC1):254:20q13 0.4561 1.00000.5439 26 31 0.0093 FGFR1:106:8p11.2-p11 FGFR2:135:10q26 UBE3A,D15S10:182:15q11- 0.4386 1.0000 0.5614 25 32 0.0117 LAMC2:18:1q25-q31MYCN(N-myc):26:2p24.1 ZNF217(ZABC1):254:20q13 0.4386 1.0000 0.5614 25 320.0117 MYCN(N-myc):26:2p24. FGFR1:106:8p11.2-p11.1 FGFR2:135:10q260.4211 1.0000 0.5789 24 33 0.0147 MYCN(N-myc):26:2p24.MYC:110:8q24.12-q24.13 FGFR2:135:10q26 0.4211 1.0000 0.5789 24 33 0.0147MYCN(N-myc):26:2p24. FGFR2:135:10q26 UBE3A, D15S10:182:15q11- 0.40351.0000 0.5965 23 34 0.0182 MYCN(N-myc):26:2p24. MYC:110:8q24.12-q24.13UBE3A, D15S10:182:15q11- 0.3860 1.0000 0.6140 22 35 0.0225FGFR2:135:10q26 UBE3A, D15S10:182:15q11 ZNF217(ZABC1):254:20q13 0.38601.0000 0.6140 22 35 0.0225 LAMC2:18:1q25-q31 FGFR1:106:8p11.2-p11.1DCC:231:18q21.3 0.5088 0.8889 0.5036 29 28 0.0260 LAMC2:18:1q25-q31MYC:110:8q24.12-q24.13 DCC:231:18q21.3 0.5088 0.8889 0.5036 29 28 0.0260LAMC2:18:1q25-q31 FGFR2:135:10q26 DCC:231:18q21.3 0.5088 0.8889 0.503629 28 0.0260 LAMC2:18:1q25-q31 UBE3A, D15S10:182:15q11 DCC:231:18q21.30.5088 0.8889 0.5036 29 28 0.0260 FGFR1:106:8p11.2-p11.MYC:110:8q24.12-q24.13 FGFR2:135:10q26 0.3684 1.0000 0.6316 21 36 0.0274MYC:110:8q24.12-q24.1 FGFR2:135:10q26 ZNF217(ZABC1):254:20q13 0.36841.0000 0.6316 21 36 0.0274 LAMC2:18:1q25-q31 MYCN(N-myc):26:2p24.1DCC:231:18q21.3 0.4912 0.8889 0.5208 28 29 0.0327 MYCN(N-myc):26:2p24.FGFR1:106:8p11.2-p11.1 MYC:110:8q24.12-q24.13 0.3509 1.0000 0.6491 20 370.0333 MYCN(N-myc):26:2p24. FGFR1:106:8p11.2-p11.1 UBE3A,D15S10:182:15q11- 0.3509 1.0000 0.6491 20 37 0.0333FGFR1:106:8p11.2-p11. MYC:110:8q24.12-q24.13 UBE3A, D15S10:182:15q11-0.3509 1.0000 0.6491 20 37 0.0333 MYCN(N-myc):26:2p24. FGFR2:135:10q26ZNF217(ZABC1):254:20q13 0.3509 1.0000 0.6491 20 37 0.0333FGFR1:106:8p11.2-p11. FGFR2:135:10q26 ZNF217(ZABC1):254:20q13 0.35091.0000 0.6491 20 37 0.0333 MYCN(N-myc):26:2p24. FGFR1:106:8p11.2-p11.1ZNF217(ZABC1):254:20q13 0.3333 1.0000 0.6667 19 38 0.0401MYCN(N-myc).26:2p24. MYC:110:8q24.12-q24.13 ZNF217(ZABC1):254:20q130.3333 1.0000 0.6667 19 38 0.0401 MYC:110:8q24.12-q24.1 UBE3A,D15S10:182:15q11 ZNF217(ZABC1):254:20q13 0.3333 1.0000 0.6667 19 380.0401 FGFR1:106:8p11.2-p11. UBE3A, D15S10:182:15q11ZNF217(ZABC1):254:20q13 0.3158 1.0000 0.6842 18 39 0.0481

Probe sets #1 and 2 in relation to aCGH data are shown in FIG. 4. Theplot illustrates that the probes were chosen in such a manner as to beable to detect both endometrioid and non-endometrioid tumors.

Fluorescence In Situ Hybridization Studies

Study Design

A probe selection study is currently being performed to determine whichfour-probe combination of the eight FISH probes can most accuratelydetect endometrial carcinoma.

Archived formalin-fixed paraffin embedded endometrial tissue specimenstaken from patients undergoing hysterectomy or Pipelle biopsy during2000 to 2006 were utilized for this probe selection study. A variety ofspecimens diagnosed as endometrial carcinoma (endometrioid type andnon-endometrioid type), simple and complex hyperplasia and normalendometrial epithelium were selected for FISH analysis (Table 4).

Ten histologically negative specimens from patients without a history ofendometrial carcinoma were also evaluated as a normal value study. Sixparaffin sectioned slides were prepared for each case, one was stainedwith hematoxylin and eosin (H&E) while five unstained slides wereprepared for FISH analysis. The H&E slide was microscopically evaluatedby a gynecologic pathologist and areas of interest (tumor in cancerspecimens; normal epithelium in benign specimens) for FISH analysis wereidentified. This area of interest was concurrently marked on theunstained tissue slide and was hybridized with each of the two FISHprobe sets (separate slides used for each of the two probe sets).

TABLE 4 Patient Population Genosensor Non Geno- cases sensor TotalNormal 0 10 10 Simple Hyperplasia 0 5 5 Complex Hyperplasia 2 4 6Endometrioid Grade 1 8 7 15 Endometrioid Grade 2 7 3 10 EndometrioidGrade 3 6 4 10 Serous 6 3 9 Clear Cell 1 1 2 MMMT 2 1 3 Total 32 38 70

Hybridized slides were evaluated using a fluorescence microscope. Thearea of interest was identified, 50 cells were evaluated (50 tumor cellsfor the cancer or pre-neoplastic cases and 50 normal cells for thenormal cases) and the number of signals from each of the four probes wasrecorded. A representative example of endometrial cancer cellsexhibiting multiple gains for these probes is shown in FIG. 5. Astatistical analysis was performed by Abbott Molecular, Inc. using thesignal patterns from all recorded cells. The ten histologically negativespecimens were used to calculate the number of signals present in normalendometrial tissue. ROC curve analyses were performed to determine theoptimal cutoff values used to discriminate chromosomal abnormalities(gains and losses) from cells with normal chromosomal content for eachprobe analyzed. Numerous different four-probe combinations wereevaluated using this technique to determine the best probe combinationto distinguish endometrial cancer from normal endometrial tissue andprecursor lesions.

Results

Probe Sets #1 and #2

Two probe sets, #1 and #2, were evaluated on all specimens. FIG. 6illustrates the proportion of cells with chromosomal abnormalities byhistologic subtype for each of the eight probes. Normal endometrialspecimens exhibited zero or very few cells with chromosomal gains andapproximate 5-20% of cells showed a form of chromosomal loss. Inadditional very few (<10%) cells with chromosomal gains were identifiedin hyperplasia specimens.

The following FISH parameters were analyzed:

-   -   1. % Gain, percent of cells with a copy number gain (>2 copies        per cell) of a locus out of 50 cells counted (50=100%)    -   2. % Loss, percent of cells with a copy number loss (<2 copies        per cell) of a locus out of 50 cells counted (50=100%)    -   3. % Abnormal, percent of cells with either a copy number loss        (<2 copies per cell) OR a copy number gain (>2 copies per cell)        of a locus out of 50 cells counted (50=100%)

The conclusions drawn from FIG. 6 are: No gains were observed in Normalspecimens for DCC, CEP18, MYC and 1q24. The increase in % cells withgains was observed from normal to hyperplasia to cancer, with most gainsin Non-Endometrioid tumors. With losses, there is no clear separationbetween normal, hyperplasia and cancer. A trend is observed in theincrease of % Abnormal from normal to hyperplasia to cancer, with thegreatest number of abnormalities in Non-Endometrioid tumors. MYC and1q24 appear to have the largest range of difference.

Probes directed to 1q25 and 8q24 had the highest percentage of abnormalcells in specimens with EN and NE cancers. All other probes detectedapproximately the same proportion of cells with gains in EN and NEcancers. For chromosomal losses, 18q (DCC) and CEP 18 exhibited the mostcells with loss in EN and NE cancers, as well as normal and hyperplasiaspecimens.

A multivariate analysis was performed to determine which four-probecombination yielded the great combination of sensitivity and specificityfor NE and NE caners. Using JMP 8.0 Statistical Analysis software (SASInstitute, 2008) 70 4-probe combinations of 8 probes were tested. “%Abnormal” was chosen as FISH parameter for analysis (see definitions)and Nominal Logistic Regression platform was utilized with the Testspecimens chosen as “all cancers” and Control specimens ashyperplasia+normal. ROCs were constructed, and 20 best combinations withthe highest AUC were selected. The combinatorial Excel program was runon the selected combinations to obtain high-resolution data onsensitivity and specificity. The program constructed contingency tablesand calculated DFI values for the 4-probe combinations at each cutofflevel of % “abnormal” for each probe.

The results of these analyses revealed numerous possible candidatefour-probe sets (Table 5). The best probe set combination included DCC,1q24, MYC, and CEP18 which had a AUC of 0.952 with a sensitivity of1.000 and a specificity of 0.9048. Three other combinations had asensitivity of 0.979 and a specificity of 0.900 which included (set 2)1q24, MYC, FGFR2, CEP18, (set 3) DCC, 1q24, FGFR2, CEP18, and (set 4)1q24, MYC, CEP18 and PIK3CA.

TABLE 5 Probe combinations that discriminate Cancer from Normal andHyperplasia. Best Best Locus Locus Locus Locus AUC Sens Spec 1 2 3 4(JMP) LM LM DCC 1q24 MYC CEP18 0.95238 1.0000 0.9048 1q24 MYC FGFR2CEP18 0.95918 0.9796 0.9000 DCC 1q24 FGFR2 CEP18 0.95867 0.9796 0.90001q24 MYC CEP18 PIK3CA 0.95306 0.9796 0.9000 DCC 1q24 MYC FGFR2 0.957140.9592 0.9000 1q24 NMYC FGFR2 CEP18 0.95714 0.9592 0.9000 1q24 FGFR2CEP18 PIK3CA 0.95714 0.9592 0.9000 1q24 FGFR2 CEP18 20q13 0.95612 0.95920.9000 1q24 CEP18 PIK3CA 20q13 0.95612 0.9592 0.9000 1q24 NMYC CEP18PIK3CA 0.9551 0.9592 0.9000 DCC 1q24 FGFR2 20q13 0.95408 0.9388 0.90001q24 MYC CEP18 0.9796 0.9048 DCC 1q24 CEP18 0.9796 0.9048 1q24 CEP180.9592 0.9048 Analyzed % Cells with any abnormality (% abnormal) usingJMP8.0 with combinatorial Excel program (contingency table analysis).Test = all cancers; Control = hyperplasia + normal

FIGS. 7 and 8 present Receiver Operator curves for the selected probecombination. It is evident from the figures that adding probes to asingle-probe FISH assay improves sensitivity and specificity.

The distribution by tumor type and grade is shown below (Table 6):

TABLE 6 Tumor Type and Grade By FISH (4Best) % Abnormal: POS or NEG.Cutoffs used as listed in FIG. 7. Count Row % NEG POS Total Normal 10 010 100.00 0.00 Simple hyperplasia 4 1 5 80.00 20.00 Complex hyperplasia5 1 6 83.33 16.67 Endometrioid Grade 1 0 15 15 0.00 100.00 EndometrioidGrade 2 0 10 10 0.00 100.00 Endometrioid Grade 3 0 10 10 0.00 100.00Carcinosarcoma/MMMT 0 3 3 0.00 100.00 Clear cell 0 2 2 0.00 100.00Serous 0 9 9 0.00 100.00 19 51 70

Detection of positives in the complex hyperplasia specimens could be dueto heterogeneity in this category and could reflect risk of progressionto cancer.

Comparison of FISH to CGH Array Data

Performance of the 1q24, CEP16, DCC and MYC FISH probe set was comparedby contingency analysis in JMP 8.0 to the aCGH Probe Selection Set #1:

DCC (18q21.3)—frequency of loss in all cancers ˜17%

Cep18—included for FISH probe set (detection of deletions)

LAMC2 (1q25)—frequency of gain in all cancers >20%

MYC (8q24.12-q24.13)—frequency of gain in all cancers ˜17%

Contingency Table Array (Ca vs N) One of Loci Changed Count Row % NEGPOS Ca/NoCa Ca 28 29 57 49.12 50.88 N 8 1 9 88.89 11.11 36 30 66Sensitivity = 50.88% (29 out of 57) Specificity = 88.89% (8 out of 9)

Contingency Table FISH (Ca vs N + Hyperplasia) FISH (4Best) % Imbal: POSor NE

Count Row % NEG POS Test or Ref? Ca 0 49 49 0.00 100.00 N + Hyperpl 19 221 90.48 9.52 19 51 70 Sensitivity = 100% (49 out of 49) Specificity =90.48% (19 out of 21)

indicates data missing or illegible when filed

It is apparent that FISH assay with probe designed based on microarrayresults has significantly improved on array performance. This ispossibly due to influence of benign cells in the macro-dissected tumorsthat dilute the analyte tumor DNA and thus lead to lower sensitivity.This problem in microarray experiments could be overcome by carefulselection of specimens with high percentage of tumor cells and bymicro-dissection of the tumor area.

Analysis of Copy Number Gains Only, Best Combinations: Cancer VsNormal+Hyperplasia

For a practical FISH application, an alternative probe combination wasevaluated that avoids technically challenging detection of losses(looses and gains are considered in “% abnormal” parameter).

Following the procedure outlined above, all 4-probe combinations wereanalyzed, and best were selected (preliminary analysis using SD of %Cells in excel), Table 7 and FIG. 9.

TABLE 7 Best 4-probe combinations, Gains only. PROBE PROBE PROBE PROBECUTOFF CUTOFF CUTOFF CUTOFF 1 2 3 4 SENS SPEC DFI 1 2 3 4 1q24 MYC CEP1820q13.2 0.84 0.95 0.17 41.30 8.36 7.91 4.46 1q24 CEP18 PIK3CA 20q13.20.84 0.95 0.17 8.36 7.91 18.47 4.46 1q24 CEP18 20q13.2 FGFR2 0.84 0.900.19 8.36 7.91 4.46 15.71

Interestingly, the combinations listed above have improved onspecificity of cancer detection but have decreased sensitivity,especially to early-stage endometrioid tumors (FIG. 9 and Table 8).

The distribution of positive and negative specimens using the 1q24, MYC,CEP18 and 20q13 probe set is shown in Table 8.

TABLE 8 Tumor Type and Grade By FISH: Gains of MYC, CEP18, 1q24, 20q13.Cutoffs used as listed in FIG. 9. Count Row % NEG POS Total Normal 10 010 100.00 0.00 Simple hyperplasia 4 1 5 80.00 20.00 Complex hyperplasia5 1 6 83.33 16.67 Endometrioid Grade 1 3 12 15 20.00 80.00 EndometrioidGrade 2 1 9 10 10.00 90.00 Endometrioid Grade 3 0 10 10 0.00 100.00Carcinosarcoma/MMMT 0 3 3 0.00 100.00 Clear cell 0 2 2 0.00 100.00Serous 0 9 9 0.00 100.00 23 47 70

As evident from the table, this set (FISH gains) has lower sensitivitytowards low-grade endometrial tumors.

Probe Set #3 Evaluation

Probe set #3 was evaluated on 16 early-stage endometrioid cancerspecimens and 12 benign (normal and hyperplasia) specimens to determinewhether it improves on sensitivity of detection of early-stage (grade 1and 2) endometrioid tumors with the FISH assay that evaluates gains ofMYC, CEP18, 1q24, 20q13.

The analysis of the FISH data has demonstrated that the addition of thePTEN loss to the selected probe set at a cutoff of 14% could increasethe sensitivity of detection of endometrial cancer to 100%, howeverdecreasing the specificity (FIG. 10). This finding is in agreement withaCGH data discussed above. In contrast, an addition of Chromosome 10 “%abnormal cells” to the selected probes at a cutoff of 10-12% of cellswith any abnormality (gain or loss), allowed for increased sensitivitywithout sacrificing the specificity. Interestingly, a combination ofCEP10 with MYC, CEP18, and 1q24 yielded the same 100% sensitivity forthe 16 cancer specimens and 92% specificity against 12normal+hyperplasia specimens. Therefore, an added assessment of aneusomy10 to the FISH probe set could be a beneficial. Addition of FGFR1 didnot significantly improve cancer detection, however, when used incombination with CEP10, 1q24 and MYC probes, FGFR1 gain at a cutoff of2% cells yielded 100% sensitivity for the 16 cancer specimens and 92%specificity against 12 normal+hyperplasia specimens. However, when allthe 70 specimens are considered (with limited data included for theprobe set #3), the only probe that did not result in decrease inspecificity when combined with the MYC, 1q24, 20q13, and CEP18 gainprobes was FGFR1 at the cutoff of ≧2% cells with gain of the probe:

Test or Ref? By MYC gain, 1q24 gain, 20q13 gain, CEP18 gain, FGFR1 gain:

Count Row % NEG POS Total Ca 2 47 49 4.08 95.92 N + Hyperpl 19 2 2190.48 9.52 21 49 70

Interestingly though, a combination of 20q13.2, 1q24, CEP10 and FGFR1 onall 70 specimens (with the data available thus far) demonstratedsensitivity and specificity of 96% and 91%, as shown below:

Test or Ref? By 20q13, CEP10, 1q24, FGFR1

Count Row % NEG POS Total Ca 2 47 49 4.08 95.92 N + Hyperpl 19 2 2190.48 9.52 21 49 70

As data for all of the specimens is unavailable at this point for ProbeSet #3, it appears feasible that the combination of 4 probes thatevaluate 20q13 gain, 1q24 gain, CEP10 imbalance, and FGFR1 gain couldprove to be superior to the 1q24, MYC, CEP8 and 20q13.2 probe set infuture experiments.

Probe Set #4 Evaluation

Probe set #4 was evaluated on the same set of endometrioid cancerspecimens, normal and hyperplasia specimens described above to determinewhether it improves on sensitivity of detection of low grade (grade 1and 2) endometrioid tumors above that obtained with the FISH assay thatevaluates gains of MYC, CEP18, 1q25, 20q13.

The analysis of the FISH data demonstrates that the substitution of theFGFR1 gain (with a cutoff of 4%) for 20q in the MYC, 1q25, and CEP probeset provided a sensitivity of 90%, which was similar to the MYC, 1q25,CEP18 and 20q probe set. However, the addition of FGFR1, resulted in oneadditional complex hyperplasia specimen to be diagnosed as positive.(Table 9).

Interestingly, further analyses revealed that FGFR1 could significantlyincrease the sensitivity of the four probe set over 20q. When the cutoffof FGFR1 was reduced to 2%, the probe combination of FGFR1, MYC, 1q25,and CEP18 had a sensitivity and specificity of 96% and 81% respectively.

TABLE 9 Evaluation of additional probes (probe set 3) to improvesensitivity of endometrial cancer detection over that obtained with MYC,CEP 18, 1q25 and 20q13. DCC/ PTEN/ FGFR1 % ID Type and MYC % CEP18 %1q25 % 20q13.2 % FGFR1 % FGFR1 % CEP18 % CEP10 % PTEN % CEP10 % Gain OR# Grade Gain >4 Gain >4 Gain >6 Gain >4 Gain >4 Gain >2 Loss >16Loss >12 Loss >18 Loss >10 Loss >10 101 N NEG NEG NEG NEG NEG NEG NEGNEG NEG NEG NEG 102 N NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS 103 NNEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG 104 N NEG NEG NEG NEG NEGNEG POS NEG NEG NEG NEG 105 N NEG NEG NEG NEG NEG NEG NEG NEG NEG NEGNEG 106 N NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG 107 N NEG NEG NEGNEG NEG NEG NEG NEG NEG NEG NEG 108 N NEG NEG NEG NEG NEG NEG NEG NEGNEG NEG NEG 109 N NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG 110 N NEGNEG NEG NEG NEG NEG NEG NEG POS POS NEG 86 SH NEG NEG NEG NEG NEG NEGPOS NEG POS POS NEG 88 SH POS POS POS NEG NEG NEG NEG NEG NEG NEG NEG 96SH NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG 97 SH NEG NEG NEG NEG NEGNEG NEG NEG NEG NEG NEG 100 SH NEG NEG NEG NEG NEG NEG NEG NEG NEG NEGNEG 85 CH POS POS NEG NEG POS POS POS POS POS POS NEG 87 CH NEG NEG NEGNEG NEG NEG NEG NEG NEG NEG NEG 89 CH NEG NEG NEG NEG NEG NEG NEG NEGNEG NEG NEG 90 CH NEG NEG NEG NEG POS POS NEG NEG NEG NEG POS 91 CH NEGNEG NEG NEG POS NEG POS NEG NEG NEG NEG 93 CH NEG NEG NEG NEG NEG NEGNEG NEG NEG NEG NEG 2 EG1 NEG NEG NEG NEG NEG NEG POS NEG POS POS POS 3EG1 POS POS POS NEG NEG NEG NEG NEG NEG POS NEG 4 EG1 POS POS POS POSPOS NEG NEG NEG NEG NEG NEG 5 EG1 POS POS POS POS POS POS NEG NEG NEGPOS NEG 14 EG1 POS POS POS POS POS POS NEG NEG NEG NEG POS 15 EG1 NEGNEG NEG POS POS NEG POS NEG NEG POS POS 16 EG1 NEG NEG NEG NEG POS POSPOS NEG POS POS NEG 18 EG1 POS POS POS NEG NEG NEG POS NEG NEG NEG NEG56 EG1 NEG NEG NEG NEG NEG NEG POS NEG POS POS NEG 57 EG1 POS NEG NEGNEG NEG NEG NEG NEG POS POS NEG 58 EG1 NEG POS NEG POS NEG POS NEG NEGNEG POS NEG 59 EG1 NEG NEG POS NEG NEG NEG NEG NEG POS POS NEG 61 EG1NEG POS NEG NEG NEG NEG POS NEG NEG POS NEG 63 EG1 POS POS POS NEG NEGNEG NEG NEG NEG NEG NEG 65 EG1 NEG POS NEG NEG NEG POS NEG NEG POS POSNEG 7 EG2 POS POS POS NEG NEG POS NEG NEG NEG NEG NEG 8 EG2 POS POS NEGNEG NEG NEG NEG NEG NEG NEG NEG 33 EG2 POS POS POS NEG NEG NEG POS NEGPOS POS NEG 34 EG2 POS NEG NEG NEG POS POS POS NEG NEG NEG POS 35 EG2POS POS POS NEG POS NEG NEG NEG NEG NEG POS 36 EG2 POS POS POS POS POSPOS NEG POS NEG NEG POS 37 EG2 POS POS POS NEG NEG NEG NEG NEG POS POSNEG 66 EG2 POS POS POS POS POS NEG POS NEG POS POS NEG 67 EG2 NEG NEGNEG NEG POS NEG NEG NEG NEG NEG NEG 70 EG2 POS POS POS NEG POS POS POSNEG POS POS POS 9 EG3 POS POS POS NEG NEG POS POS NEG POS POS NEG 42 EG3POS POS POS POS POS NEG POS NEG NEG NEG POS 43 EG3 POS POS POS POS POSPOS POS NEG NEG NEG POS 44 EG3 POS POS POS NEG POS NEG NEG NEG NEG NEGNEG 45 EG3 POS POS POS POS POS POS NEG NEG NEG NEG POS 47 EG3 POS POSPOS POS POS POS POS POS POS NEG POS 75 EG3 POS POS POS POS POS POS NEGNEG NEG NEG POS 76 EG3 POS NEG POS POS NEG POS POS POS POS POS POS 77EG3 POS POS POS POS POS POS NEG POS NEG NEG POS 83 EG3 POS POS POS POSPOS POS POS NEG NEG POS POS 11 C/M POS NEG NEG POS POS POS NEG POS POSPOS POS 12 C/M POS POS POS POS POS POS POS POS POS POS POS 78 C/M POSPOS POS POS POS POS POS POS NEG NEG POS 54 Clear POS POS POS POS POS POSNEG POS NEG NEG POS cell 81 Clear POS POS POS POS POS POS POS POS NEGNEG POS cell 1 S POS POS POS POS POS POS POS POS NEG POS POS 6 S POS POSPOS POS POS POS POS POS NEG NEG POS 10 S POS POS POS POS POS POS POS POSNEG NEG POS 49 S POS POS POS POS POS POS POS POS NEG NEG POS 50 S POSPOS POS POS POS POS NEG NEG NEG NEG POS 51 S POS POS POS POS POS POS POSPOS NEG NEG POS 79 S POS POS POS POS POS POS POS POS NEG NEG POS 80 SPOS POS POS POS POS POS POS POS NEG NEG POS 94 S POS POS POS POS POS POSPOS POS NEG POS POS Table Abbreviations: N = Normal; SH = Simplehyperplasia; CH = Complex hyperplasia; EG1 = Endometrioid Grade 1; EG2 =Endometrioid Grade 2; EG3 = Endometrioid Grade 3; Carcinosarcoma/MMMT =CM; S = Serous; POS = Number of cells with abnormal sign patternsidentified met or exceeded the threshold for the probe; NEG = Number ofcells with abnormal sign patterns identified was less than the thresholdfor the probe

Analyses of other probes to increase the sensitivity of MYC, 1q25 andCEP18 are shown in Table 10. The addition of DCC, CEP10, PTEN, or FGFR1increase the sensitivity of the combination probe set to 94-98%.However, the increase in sensitivity was achieved at the expense ofdecreased specificity (76-81%).

TABLE 10 Analysis of adding DCC, CEP10, PTEN or FGFR1 to a probe set ofMYC, 1q25 and CEP18 to increase the sensitivity of endometrial cancerdetection Gains Losses Sensi- Speci- Probe 1 Probe 2 Probe 3 Probe 4tivity ficity Cut- MYC >4 1q25 >6 CEP18 >4 DCC/ 98% 76% off CEP18 >16Cut- MYC >4 1q25 >6 CEP18 >4 CEP10 >10 98% 81% off Cut- MYC >4 1q25 >6CEP18 >4 PTEN >18 96% 81% off Cut- MYC >4 1q25 >6 CEP18 >4 FGFR1 >10 94%81% off (gains and loss)

Additional analyses were performed to determine the optimal probe setwith cutoffs to attain very high specificity for endometrial carcinoma.Results from this analysis can be seen in Table 11. Numerous probe andabnormality combinations achieved at least 95% specificity. Those thatproduced the highest sensitivity with greater than 95% specificity arehighlighted in yellow at the top of Table 11. All of these combinationsincluded the evaluation of locus loss or combination of loss and gains(imbal). The best performing probe set that only evaluated gains had asensitivity of 84% and specificity of 95%, which included FGFR1, 1q25,CEP18 and 20q13 with cutoffs of 8%, 8%, 8% and 8% respectively(highlighted in blue). Two other probe sets containing 1q25, MYC, CEP18and FGFR1 or 20q13 using cutoffs of 12%, 12%, 12%, and 12%,respectively, achieved a sensitivity of 80% and specificity of 95%(highlighted in green).

The most appealing probe set included 4 LSI probes of 1q25, MYC, FGFR1and 20q13 using similar cutoffs also had a sensitivity and specificityof 80% and 95%, respectively (seen in red). This probe set is ideal duebecause it performs nearly as well as other probe sets while onlyanalyzing specimens for chromosomal gains. Chromosomal gains are easierto identify by technologists and would likely have a higherinter-observer reproducibility than when evaluating chromosomal losses.

TABLE 11 Analysis of different probes and cutoffs to achieve specificityof >95% for endometrial cancer detection PROBE 1 PROBE 2 PROBE 3 PROBE 4Cut Cut Cut Cut Probe Abn off Probe Abn off Probe Abn off Probe Abn offSENS SPEC PTEN/ loss 12 FGFR1 imbal 12 1q25 gain 12 MYC gain 12 0.8570.952 CEP10 FGFR1 imbal 12 FGFR1 loss 12 1q25 gain 12 MYC gain 12 0.8570.952 FGFR1 imbal 12 FGFR1 gain 12 1q25 gain 12 MYC gain 12 0.857 0.952FGFR1 imbal 12 1q25 gain 12 MYC gain 12 CEP18 gain 12 0.857 0.952 FGFR1gain 8 1q25 gain 8 CEP18 gain 8 20q13.2 gain  8 0.837 0.952

PTEN/ loss 12 FGFR1 imbal 12 1q25 gain 12 CEP18 gain 12 0.816 0.952CEP10 FGFR1 loss 12 1q25 gain 12 MYC gain 12 CEP18 gain 12 0.816 0.952PTEN/ loss 12 FGFR1 gain 12 1q25 gain 12 MYC gain 12 0.796 0.952 CEP10FGFR1 gain 28 CEP10 imbal 28 1q25 gain 28 MYC gain 28 0.796 0.952 PTEN/loss 12 1q25 gain 12 MYC gain 12 CEP18 gain 12 0.796 0.952 CEP10

PTEN/ loss 12 1q25 gain 12 MYC gain 12 20q13.2 gain 12 0.796 0.952 CEP10

Abbreviations: Probe = locus of interest; Abn = Type of abnormality;loss = evaluates only loss of locus; gain = evaluates only gain oflocus; imbal = evaluates gains or loss; cutoff = % of cells withabnormality to consider a specimen as positive.

ATTACHMENT 1. GeneSensor ™ 300 Array Clone List Cyto Loc, Locus LinkUtility 1 CEB108/T7 1p tel Sub Tel 2 1PTEL06 1p tel Sub Tel 3CDC2L1(p58) 1p36 u DEL 4 PRKCZ 1p36.33 u DEL 5 TP73 1p36.33 u DEL/LOH 6D1S2660 1p36.32 u DEL/LOH 7 D1S214 1p36.31 u DEL/LOH 8 D1S1635 1p36.22LOH 9 D1S199 1p36.13 LOH 10 FGR(SRC2) 1p36.2-p36.1 AMP(1) 11 MYCL1(LMYC)1p34.3 AMP(1) 12 D1S427, FAF1 1p32.3 LOH 13 D1S500 1p31.1 LOH 14 D1S4181p13.1 LOH 15 NRAS 1p13.2 AMP(1) 16 D1S2465, D1S3402 1p12 17 WI-5663,WI-13414 1q21 18 LAMC2 1q25-q31 AMP(1) 19 PTGS2(COX2) 1q31.1 20 TGFB21q41 21 AKT3 1q44 AMP 22 SHGC-18290 1q tel Sub Tel 23 1QTEL10 1q tel SubTel 24 U32389 2p tel Sub Tel 25 2PTEL27 2p tel Sub Tel 26 MYCN(N-myc)2p24.1 AMP(1) 27 MSH2, KCNK12 2p22.3-2p22.1 LOH 28 REL 2p13-p12 AMP(1)29 GNLY 2p12-q11 M 30 SGC34236 2q13 M 31 BIN1 2q14 pTSG 32 LRP1B 2q21.2pTSG 33 TBR1 2q23-q37 M 34 ITGA4 2q31-q32 M 35 CASP8 2q33-q34 LOH 36ERBB4(HER-4) 2q33.3-q34 HER-2 homol 37 WI-6310 2q tel Sub Tel 38 D2S4472q tel Sub Tel 39 3PTEL25 3p tel Sub Tel 40 3PTEL01, CHL1 3p tel Sub Tel41 VHL 3p25-p26 TSG 42 RAF1 3p25 AMP(1) 43 THRB 3p24.3 LOH 44 MLH13p21.3-p23 Del 45 RASSF1 3p21.3 pTSG 46 FHIT 3p14.2 pTSG 47 p44S103p14.1 48 D3S1274, ROBO1 3p12-3p13 LOH 49 RBP1, RBP2 3q21-q22 50 TERC3q26 AMP(1) 51 EIF5A2 3q26.2 52 PIK3CA 3q26.3 AMP(1) 53 TP63 3q27-q29TSG 54 MFI2 3q tel Sub Tel 55 3QTEL05 3q tel Sub Tel 56 GS10K2/T7 4p telSub Tel 57 SHGC4-207 4p tel Sub Tel 58 D4S114 4p16.3 u DEL 59 WHSC14p16.3 u DEL 60 DDX15 4p15.3 M 61 KIT 4q11-q12 ONC 62 PDGFRA 4q11-q13AMP(1) 63 EIF4E 4q24 (by AMP ucsc) 64 PGRMC2 4q26 65 PDZ-GEF1 4q32.1 M66 4QTEL11 4q tel Sub Tel 67 D4S2930 4q tel Sub Tel 68 C84C11/T3 5p telSub Tel 69 D5S23 5p15.2 u DEL 70 D5S2064 5p15.2 u DEL 71 DAB2 5p13 pTSG72 DHFR, MSH3 5q11.2-q13.2 gain/loss Ca 73 APC 5q21-q22 Del 74 EGR15q31.1 Del 75 CSF1R 5q33-q35 Del 76 NIB1408 5q tel Sub Tel 77 5QTEL70 5qtel Sub Tel 78 6PTEL48 6p tel Sub Tel 79 PIM1 6p21.2 M 80 CCND3 6p21 AMP81 D6S414 6p12.1-p21.1 82 HTR1B 6q13 M 83 D6S434 6q16.3 Del 84 D6S2686q16.3-q21 LOH 85 MYB 6q22-q23 AMP(1) 86 D6S311 6q23-24 LOH 87 ESR16q25.1 AMP(1) 88 6QTEL54 6q tel Sub Tel 89 G31341 7p tel Sub Tel 90 IL67p21 M 91 EGFR 7p12.3-p12.1 AMP(1) 92 ELN 7q11.23 u DEL 93 RFC2, CYLN27q11.23 u DEL 94 ABCB1(MDR1) 7q21.1 AMP(1) 95 CDK6 7q21-q22 AMP 96SERPINE1 7q21.3-q22 pTSG 97 MET 7q31 AMP(1) 98 TIF1 7q32-q34 M 99stSG48460 7q tel Sub Tel 100 7QTEL20 7q tel Sub Tel 101 D8S504 8p telSub Tel 102 D8S596 8p tel Sub Tel 103 CTSB 8p22 AMP(1) 104 PDGRL8p22-p21.3 Del 105 LPL 8p22 Del 106 FGFR1 8p11.2-p11.1 AMP(1) 107 MOS8q11 AMP(1) 108 E2F5 8p22-q21.3 M 109 EXT1 8q24.11-q24.13 TSG, uDel 110MYC 8q24.12-q24.13 AMP(1) 111 PTK2 8q24-qter AMP 112 SHGC-31110 8q telSub Tel 113 U11829 8q tel Sub Tel 114 AF170276 9p tel Sub Tel 115 D9S9139ptel Sub Tel 116 MTAP 9p21.3 LOH 117 CDKN2A(p16), MTAP 9p21 TSG 118AFM137XA11 9p11.2 M 119 D9S166 9p12-q21 120 PTCH 9q22.3 TSG 121 DBCCR19q33.2 TSG 122 TSC1 9q34 TSG 123 ABL1 9q34.1 AMP(1) 124 H18962 9q telSub Tel 125 D9S325 9q tel Sub Tel 126 10PTEL006 10p tel Sub Tel 127SHGC-44253 10p tel Sub Tel 128 D10S249, D10S533 10p15 TSG 129 GATA310p15 130 WI-2389, D10S1260 10p14-p13 u DEL 131 BMI1 10p13 gain 132D10S167 10p11-10q11 near Cen 133 EGR2 10q21.3 M 134 PTEN 10q23.3 TSG 135FGFR2 10q26 AMP(1) 136 DMBT1 10q25.3-q26.1 137 stSG27915 10q tel Sub Tel138 10QTEL24 10q tel Sub Tel 139 11PTEL03 11p tel Sub Tel 140 INS 11ptel Sub Tel 141 HRAS 11p15.5 AMP(1) 142 CDKN1C(p57) 11p15.5 TSG 143 WT111p13 TSG 144 KAI1 11p11.2 145 D11S461 11q12.2 near cen 146 MEN1 11q13147 CCND1 11q13 AMP(1) 148 FGF4, FGF3 11q13 AMP(1) 149 EMS1 11q13 AMP(1)150 GARP 11q13.5-q14 AMP(1) 151 PAK1 11q13-q14 AMP(1) 152 RDX 11q22.3LOH 153 ATM 11q22.3 LOH 154 MLL 11q23 AMP(1) 155 WI-6509 11q tel Sub Tel156 AF240622 11q tel Sub Tel 157 8M16/SP6 12p tel Sub Tel 158 SHGC-555712p tel Sub Tel 159 CCND2 12p13 AMP(1) 160 CDKN1B(p27) 12p13.1-p12 TSG161 KRAS2 12p11.2 AMP(1) 162 WNT1(INT1) 12q12-q13 AMP(1) 163 CDK2, ERBB312q13 AMP 164 GLI 12q13.2-q13.3 AMP(1) 165 SAS, CDK4 12q13-q14 AMP(1)166 MDM2 12q14.3-q15 AMP(1) 167 DRIM, ARL1 12q23 168 stSG8935 12q telSub Tel 169 U11838 12q tel Sub Tel 170 BRCA2 13q12-q13 TSG 171 RB1 13q14TSG 172 D13S319 13q14.2 LOH 173 D13S25 13q14.3 LOH 174 D13S327 13q telSub Tel 175 PNN(DRS) 14q13 176 TCL1A 14q32.1 gain/loss 177 AKT1 14q32.32AMP(1) 178 IGH(D14S308) 14q tel Sub Tel 179 IGH(SHGC-36156) 14q tel SubTel 180 D15S11 15q11-q13 u DEL 181 SNRPN 15q12 u DEL 182 UBE3A, D15S1015q11-q13 u DEL 183 GABRB3 15q11.2-q12 u DEL 184 MAP2K5 15q23 185 FES15q26.1 AMP(1) 186 IGF1R 15q25-q26 AMP(1) 187 PACE4C 15q tel Sub Tel 188WI-5214 15q tel Sub Tel 189 16PTEL03 16p tel Sub Tel 190 stSG48414 16ptel Sub Tel 191 CREBBP 16p13.3 u DEL 192 EMP2 16p13.3 193 ABCC1(MRP1)16p13.1 AMP(1) 194 CYLD 16q12-q13 TSG 195 CDH1 16q22.1 LOH 196 FRA16D16q23.2 197 CDH13 16q24.2-q24.3 LOH 198 LZ16 16q24.2 Del 199 FANCA16q24.3 200 stSG30213 16q tel Sub Tel 201 16QTEL013 16q tel Sub Tel 202282M15/SP6 17p tel Sub Tel 203 WI-14673 17p tel Sub Tel 204 HIC1 17p13.3LOH 205 D17S379, MNT 17p13.3 u DEL/LOH 206 PAFAH1B1(LIS1) 17p13.3 u DEL207 TP53(p53) 17p13.1 TSG 208 D17S125, D17S61 17p12-p11.2 u Del/u Dup209 D17S1296, D17S1523 17p12-p11.2 u Del/u Dup 210 LLGL1 17p12-17p11.2 uDEL 211 FLI, TOP3A 17p12-17p11.2 u DEL 212 NF1 5′ 17q11.2 213 NF1 3′17q11.2 214 BRCA1 17q21 TSG 215 PPARBP(PBP) 17q12 AMP 216 ERBB2(HER-2)17q11.2-17q12 AMP(1) 217 THRA 17q11.2 AMP 218 TOP2A 17q21-q22 AMP 219NME1(NME23) 17q21.3 LOH? 220 RPS6KB1(STK14A) 17q23 AMP(1) 221 D17S167017q23 222 TK1 17q23.2-q25.3 AMP? 223 SHGC-103396 17q tel Sub Tel 224AFM217YD10 17q tel Sub Tel 225 D18S552 18p tel Sub Tel 226 SHGC17327 18ptel Sub Tel 227 YES1 18p11.31-p11.21 AMP(1) 228 TYMS(TS) 18p11.32 AMP229 LAMA3 18q11.2 230 FRA18A(D18S978) 18q12.3 231 DCC 18q21.3 Del 232MADH4(DPC4) 18q21.1 TSG 233 BCL2 3′ 18q21.3 AMP 234 CTDP1, SHGC-14582018q tel Sub Tel 235 18QTEL11 18q tel Sub Tel 236 129F16/SP6 19p tel SubTel 237 stSG42796 19p tel Sub Tel 238 INSR 19p13.2 AMP(1) 239 JUNB19p13.2 AMP(1) 240 CCNE1 19q12 AMP(1) 241 AKT2 19q13.1-q13.2 LOH 242GLTSCR2, SULT2A1 19q13.32 243 D19S238E 19 q tel Sub Tel 244 20PTEL18 20ptel Sub Tel 245 SOX22 20p tel Sub Tel 246 JAG1 20p12.1-p11.23 uDel 247MKKS, SHGC-79896 20p12.1-p11.23 uDel 248 TOP1 20q12-q13.1 AMP(1) 249NCOA3(AIB1) 20q12 AMP(1) 250 MYBL2 20q13.1 AMP(1) 251 CSE1L(CAS) 20q13AMP(1) 252 PTPN1 20q13.1-q13.2 AMP(1) 253 STK6(STK15) 20q13.2-q13.3AMP(1) 254 ZNF217(ZABC1) 20q13.2 AMP(1) 255 CYP24 20q13.2 AMP 256TNFRSF6B(DCR3) 20q13 AMP 257 TPD52L2, TOM 20q tel Sub Tel 258 20QTEL1420q tel Sub Tel 259 D21S378 21q11.2 M 260 RUNX1(AML1) 21q22.3 AMP(1) 261DYRK1A 21q22 gain 262 D21S341, D21S342 21q22.3 gain 263 PCNT2(KEN) 21qtel Sub Tel 264 21QTEL08 21q tel Sub Tel 265 D22S543 22q11 M 266 GSCL22q11.21 u DEL 267 HIRA(TUPLE1) 22q11.21 u DEL 268 TBX1 22q11.2 u DEL269 BCR 22q11.23 AMP(1) 270 NF2 22q12.2 TSG 271 PDGFB(SIS) 22q13.1AMP(1) 272 ARHGAP8 22q13.3 273 ARSA 22q tel Sub Tel 274 22QTEL31 22q telSub Tel 275 DXYS129 X/Yp tel Sub Tel 276 STS 3′ Xp22.3 u DEL 277 STS 5′Xp22.3 u DEL 278 KAL Xp22.3 u DEL 279 DMD exon 45-51 Xp21.1 280 DXS580Xp11.2 281 DXS7132 Xq12 282 AR 3′ Xq11-q12 AMP(1) 283 XIST Xq13.2 284OCRL1 Xq25 285 EST CDY16c07 X/Yq tel Sub Tel 286 SRY Yp11.3 287 AZFaregion Yq11 Sub Tel Single copy sequence near the telomere AMP(1) CancerAmplicon previously placed on AmpliOncl Chip AMP Cancer Amplicon notpreviously placed on AmpliOncl Chip u DEL Region lost in microdeletionsyndrome TSG Tumor Supressor Gene pTSG putqtive Tumor Supressor Gene MMarker added to reduce genomic gaps gain Region gained in cancer u DupMicro Duplication LOH Region of Loss of Heterozygosity Del DeletionRegion near Cen Single copy sequence near the centromere

1. A method of detecting the presence of endometrial carcinoma in abiological sample from a subject, the method comprising: contacting thesample with one or more probes for one or more chromosome regionsselected from the group consisting of: 1q, 2p, 2q, 3p, 3q, 7p, 8p, 8q,9p, 9q, the centromeric region of chromosome 10, 10q, 15q, 16q, 17p, thecentromeric region of chromosome 18, 18q, 19p, 20q, and 22q; incubatingthe one or more probes with the sample under conditions in which eachprobe binds selectively with a polynucleotide sequence on its targetchromosome or chromosomal region to form a stable hybridization complex;and detecting hybridization of the one or more probes, wherein ahybridization pattern showing at least one gain or loss or imbalance ata chromosomal region targeted by the probes is indicative of endometrialcarcinoma.
 2. The method of claim 1, wherein a hybridization patternshowing a gain in one or more chromosome regions selected from the groupconsisting of: 1q, 2p, 3q, 8q, 10q, and 20q is indicative of endometrialcarcinoma.
 3. The method of claim 2, wherein a hybridization patternshowing a gain in one or more chromosome regions selected from the groupconsisting of: 1q, 10p, and 10q is indicative of endometrioid carcinoma.4. The method of claim 2, wherein a hybridization pattern showing a gainin one or more chromosome regions selected from the group consisting of:3q, 8q, 18q, and 20q is indicative of non-endometrioid carcinoma.
 5. Themethod of claim 1, wherein a hybridization pattern showing a gain in1q31-qtel is indicative of endometrial carcinoma.
 6. The method of claim1, wherein a hybridization pattern showing a loss in one or morechromosome regions selected from the group consisting of: 9p, 9q, 15q,16q, 17p, 18q, 19p, and 22q is indicative of endometrial carcinoma. 7.The method of claim 1, wherein a hybridization pattern showing a loss inone or more chromosome regions selected from the group consisting of:15q11-q13, 18q21, and 19 ptel is indicative of endometrial carcinoma. 8.The method of claim 1, wherein said one or more probes are for one ormore chromosome subregions selected from the group consisting of: 1q25,2p24, 2q26, 3p21, 3q27-q29, 7p21, 8p11, 8q24, 9q34, the centromericregion of chromosome 10, 10q23, 10q26, 15q11-q13, 16q24, the centromericregion of chromosome 18, 18q21, 20q12 and 20q13.
 9. The method of claim8, wherein said one or more probes are for one or more chromosomesubregions selected from the group consisting of: 1q25-q31, 2p24,3p21.3, 3q27-q29, 7p21, 8p11.2-p11.1, 8q24, 9q34, 10q23.3, 10q26,15q11-q13, 16q24.3, 18q21.3, 20q12, and 20q13.2.
 10. The method of claim8, wherein said one or more probes are for one or more chromosomesubregions selected from the group consisting of: 1q25, 10q23.3,18q21.2, CEP10, CEP18, 8p11.2, 8q24, 2p24.3, 2q26.32, 10q26.13, and20q13.2.
 11. The method of claim 8, wherein the sample is contacted witha combination of at least 3 probes for a set of chromosome subregionsselected from the group consisting of: 1q25, 8q24, 15q11-q13; 1q25,10q26, 15q11-q13; 1q25, 2p24, 8q24 1q25, 8q24, 10q26; 1q25, 8p11,15q11-q13; 1q25, 2p24, 8p11; 1q25, 8p11, 10q26; 1q25, 8p11, 8q24; 1q25,2p24, 10q26; 1q25, 2p24, 15q11-q13; 8q24, 10q26, 15q11-q13; 1q25, 8p11,20q13; 1q25, 8q24, 20q13; 1q25, 15q11-q13, 20q13; 1q25, 10q26, 20q13;8p11, 10q26, 15q11-q13; 1q25, 2p24, 20q13; 2p24, 8p11, 10q26; 2p24,8q24, 10q26; 2p24, 10q26, 15q11-q13; 2p24, 8q24, 15q11-q13; 10q26,15q11-q13, 20q13; 1q25, 8p11, 18q21; 1q25, 8q24, 18q21; 1q25, 10q26,18q21; 1q25, 15q11-q13, 18q21; 8p11, 8q24, 10q26; 8q24, 10q26, 20q13;1q25, 2p24, 18q21; 2p24, 8p11, 8q24; 2p24, 8p11, 15q11-q13; 8p11, 8q24,15q11-q13; 2p24, 10q26, 20q13; 8p11, 10q26, 20q13; 2p24, 8p11, 20q13;2p24, 8q24, 20q13; 8q24, 15q11-q13, 20q13; and 8p11, 15q11-q13, 20q13.12. The method of claim 11, wherein the sample is contacted with acombination of at least 3 probes for a set of chromosome subregionsselected from the group consisting of: 1q25, 18q21, CEP18, 8q24; 2p24,2q26, 10q26, 2q13; and 10q23, CEP10, and 8p11.
 13. The method of claim11, wherein the sample is contacted with a combination of at least 2probes for a set of chromosome subregions selected from the groupconsisting of: 18q21, 1q24, 8q24, CEP18; 1q24, 8q24, 10q26, CEP18;18q21, 1q24, 10q26, CEP18; 1q24, 8q24, CEP18, 3q27-q29; 18q21, 1q24,8q24, 10q26; 1q24, 2p24, 10q26, CEP18; 1q24, 10q26, CEP18, 3q27-q29;1q24, 10q26, CEP18, 20q13; 1q24, CEP18, 3q27-q29, 20q13; 1q24, 2p24,CEP18, 3q27-q29; 18q21, 1q24, 10q26, 20q13; 1q24, 8q24, CEP18; 18q21,1q24, CEP18; and 1q24, CEP18.
 14. The method of claim 11, wherein thesample is contacted with a combination of at least 4 probes for a set ofchromosome subregions selected from the group consisting of: 1q24, 8q24,CEP18, 20q13; 1q24, CEP18, 3q27-q29, 20q13; 1q24, CEP18, 20q13, 10q26;CEP10, 8q24, CEP18, 1q24; 10q26, CEP10, 1q24, 8q24; 8q24, 1q24, 20q13,CEP18; 10q26; 20q13, CEP10, 1q24, 10q26. wherein a hybridization patternshowing a gain in one or more of these chromosome subregions isindicative of endometrial carcinoma.
 15. The method of claim 14, whereinone or more of a gain at one of more of 1q24, 8q24, CEP18, and 20q13 areindicative of endometrial carcinoma.
 16. The method of claim 14, whereinone or more of a 20q13 gain, a 1q24 gain, a CEP10 imbalance, and a 10q26gain are indicative of endometrial carcinoma.
 17. The method of claim 1,wherein the sample is contacted with a combination of at least 2 probesfor a set of chromosome subregions selected from the group consistingof: 18q21, 1q25, 8q24, CEP18; 1q25, 8q24, 10q26, CEP18; 18q21, 1q25,10q26, CEP18; 1q25, 8q24, CEP18, 3q27-q29; 18q21, 1q25, 8q24, 10q26;1q25, 2p24, 10q26, CEP18; 1q25, 10q26, CEP18, 3q27-q29; 1q25, 10q26,CEP18, 20q13; 1q25, CEP18, 3q27-q29, 20q13; 1q25, 2p24, CEP18, 3q27-q29;18q21, 1q25, 10q26, 20q13; 1q25, 8q24, CEP18; 18q21, 1q25, CEP18; and1q25, CEP18.
 18. The method of claim 1, wherein the sample is contactedwith a combination of at least 4 probes for a set of chromosomesubregions selected from the group consisting of: 1q25, 8q24, CEP18,20q13; 1q25, CEP18, 3q27-q29, 20q13; 1q25, CEP18, 20q13, 10q26; CEP10,8q24, CEP18, 1q25; 10q26, CEP10, 1q25, 8q24; 8q24, 1q25, 20q13, CEP18;10q26; 20q13, CEP10, 1q25, 10q26. wherein a hybridization patternshowing a gain in one or more of these chromosome subregions isindicative of endometrial carcinoma.
 19. The method of claim 18, whereinone or more of a gain at one of more of 1q25, 8q24, CEP18, and 20q13 areindicative of endometrial carcinoma.
 20. The method of claim 18, whereinone or more of a 20q13 gain, a 1q25 gain, a CEP10 imbalance, and a 10q26gain are indicative of endometrial carcinoma.
 21. The method of claim 1wherein the probe combination distinguishes samples comprisingendometrial carcinoma from samples that do not comprise endometrialcarcinoma with a sensitivity of at least 93% and a specificity of atleast 90%.
 22. The method of claim 21, wherein the sensitivity is atleast 95% and the specificity is at least 90.4%.
 23. The method of claim22, wherein the sensitivity is least 96% and the specificity is at least91%.
 24. The method of claim 1, wherein the probe combination comprisesbetween 2 and 10 probes.
 25. The method of claim 1, wherein the probecombination comprises between 3 and 8 probes.
 26. The method of claim 1,wherein the probe combination comprises 4 probes.
 27. The method ofclaim 1, wherein the method is carried out by array comparative genomichybridization (aCGH) to probes immobilized on a substrate.
 28. Themethod of claim 1, wherein the method is carried out by fluorescence insitu hybridization, and each probe in the probe combination is labeledwith a different fluorophore.
 29. The method of claim 1, wherein thesample comprises an endometrial brushing specimen or an endometrialbiopsy specimen.
 30. The method of claim 1, wherein, when the results ofthe method indicate endometrial carcinoma, the method additionallycomprises treating the subject for endometrial carcinoma.
 31. Acombination of probes comprising between 2 and 10 probes selected fromthe groups set forth in claim 1, wherein the combination of probes has asensitivity of at least 93% and a specificity of at least 90% fordistinguishing samples comprising endometrial carcinoma from samplesthat do not comprise endometrial carcinoma. 32-35. (canceled)
 36. A kitfor diagnosing endometrial carcinoma, wherein the kit comprises acombination of probes comprising between 2 and 10 probes selected fromthe groups set forth in claim 1, wherein the combination of probes has asensitivity of at least 93% and a specificity of at least 90% fordistinguishing samples comprising endometrial carcinoma from samplesthat do not comprise endometrial carcinoma. 37-43. (canceled)